U.S. patent application number 13/861134 was filed with the patent office on 2013-08-15 for cholinergic enhancers with improved blood-brain barrier permeability for the treatment of diseases accompanied by cognitive impairment.
This patent application is currently assigned to GALANTOS PHARMA GMBH. The applicant listed for this patent is GALANTOS PHARMA GMBH. Invention is credited to ALFRED MAELICKE.
Application Number | 20130210808 13/861134 |
Document ID | / |
Family ID | 41133822 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130210808 |
Kind Code |
A1 |
MAELICKE; ALFRED |
August 15, 2013 |
CHOLINERGIC ENHANCERS WITH IMPROVED BLOOD-BRAIN BARRIER
PERMEABILITY FOR THE TREATMENT OF DISEASES ACCOMPANIED BY COGNITIVE
IMPAIRMENT
Abstract
The present invention refers to compounds that, in addition to
enhancing the sensitivity to acetylcholine and choline, and their
exogenous agonists, of neuronal cholinergic receptors and/or acting
as cholinesterase inhibitors and/or neuroprotective agents, have
enhanced blood-brain barrier permeability in comparison to their
parent compounds. The compounds are derived (either formally by
their chemical structure or directly by chemical synthesis) from
natural compounds belonging to the class of amaryllidaceae
alkaloids e.g., galantamine, narwedine and lycoramine, or from
metabolites of said compounds. The compounds of the present
invention can either interact as such with their target molecules,
or they can act as "pro-drugs", in the sense that after reaching
their target regions in the body they are converted by hydrolysis
or enzymatic attack to the original parent compound and react as
such with their target molecules, or both. The compounds of this
invention may be used as medicaments.
Inventors: |
MAELICKE; ALFRED;
(NIEDER-OLM, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GALANTOS PHARMA GMBH; |
|
|
US |
|
|
Assignee: |
GALANTOS PHARMA GMBH
Mainz
DE
|
Family ID: |
41133822 |
Appl. No.: |
13/861134 |
Filed: |
April 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12422901 |
Apr 13, 2009 |
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13861134 |
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12067799 |
Jul 2, 2008 |
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PCT/EP2006/009220 |
Sep 22, 2006 |
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12422901 |
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11683148 |
Mar 7, 2007 |
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12422901 |
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60780243 |
Mar 7, 2006 |
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60780243 |
Mar 7, 2006 |
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61046683 |
Apr 21, 2008 |
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Current U.S.
Class: |
514/215 |
Current CPC
Class: |
C07D 491/06 20130101;
C07D 307/91 20130101; C07F 9/6561 20130101; C07D 405/12 20130101;
Y02A 50/465 20180101; A61K 31/55 20130101; Y02A 50/30 20180101 |
Class at
Publication: |
514/215 |
International
Class: |
A61K 31/55 20060101
A61K031/55 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2005 |
EP |
05 020 721.6 |
Claims
1. A method for the treatment of a neurodegenerative or psychiatric
or neurological disease associated with a cholinergic deficit
comprising administering a pharmaceutical composition comprising
pro-drug compound GLN-1062 ##STR00097## or a pharmaceutically
acceptable salt thereof to a patient in need thereof.
2. The method of claim 1, wherein as a result of endogenous
enzymatic activity the pro-drug compound GLN-1062 is cleaved after
administration to produce the effective agent galantamine.
3. The method of claim 2, wherein cleavage of the pro-drug compound
GLN-1062 to produce the effective agent galantamine occurs in the
brain of a treated patient.
4. The method of claim 1, wherein the disease is selected from
Alzheimer's disease, Parkinson's disease, other types of dementia,
schizophrenia, epilepsy, stroke, poliomyelitis, neuritis, myopathy,
oxygen and nutrient deficiencies in the brain after hypoxia,
anoxia, asphyxia, cardiac arrest, chronic fatigue syndrome, various
types of poisoning, anesthesia, particularly neuroleptic
anesthesia, spinal cord disorders, inflammation, particularly
central inflammatory disorders, postoperative delirium and/or
subsyndronal postoperative delirium, neuropathic pain, subsequences
of the abuse of alcohol and drugs, addictive alcohol and nicotine
craving, and subsequences of radiotherapy.
5. The method of claim 2, wherein the disease is selected from
Alzheimer's disease, Parkinson's disease, other types of dementia,
schizophrenia, epilepsy, stroke, poliomyelitis, neuritis, myopathy,
oxygen and nutrient deficiencies in the brain after hypoxia,
anoxia, asphyxia, cardiac arrest, chronic fatigue syndrome, various
types of poisoning, anesthesia, particularly neuroleptic
anesthesia, spinal cord disorders, inflammation, particularly
central inflammatory disorders, postoperative delirium and/or
subsyndronal postoperative delirium, neuropathic pain, subsequences
of the abuse of alcohol and drugs, addictive alcohol and nicotine
craving, and subsequences of radiotherapy.
6. The method of claim 3, wherein the disease is selected from
Alzheimer's disease, Parkinson's disease, other types of dementia,
schizophrenia, epilepsy, stroke, poliomyelitis, neuritis, myopathy,
oxygen and nutrient deficiencies in the brain after hypoxia,
anoxia, asphyxia, cardiac arrest, chronic fatigue syndrome, various
types of poisoning, anesthesia, particularly neuroleptic
anesthesia, spinal cord disorders, inflammation, particularly
central inflammatory disorders, postoperative delirium and/or
subsyndronal postoperative delirium, neuropathic pain, subsequences
of the abuse of alcohol and drugs, addictive alcohol and nicotine
craving, and subsequences of radiotherapy.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The disclosure refers to compounds that, in addition to
enhancing the sensitivity to acetylcholine and choline, and to
their agonists, of neuronal cholinergic receptors, and/or acting as
cholinesterase inhibitors and/or neuroprotective agents, have
enhanced blood-brain barrier permeability in comparison to their
parent compounds. The compounds are derived (either formally by
their chemical structure or directly by chemical synthesis) from
natural compounds belonging to the class of amaryllidaceae
alkaloids e.g., Galantamine, Narwedine and Lycoramine, or from
metabolites of said compounds. The compounds of the present
invention can either interact as such with their target molecules,
or they can act as "pro-drugs", in the sense that after reaching
their target regions in the body, they are converted by hydrolysis
or enzymatic attack to the original parent compound and react as
such with their target molecules, or both. The compounds of this
disclosure may be used as medicaments for the treatment of human
brain diseases associated with a cholinergic deficit, including the
neurodegenerative diseases Alzheimer's and Parkinson's disease and
the neurological/psychiatric diseases vascular dementia,
schizophrenia and epilepsy. Galantamine derivatives disclosed
herein have higher efficacy and lower levels of adverse side
effects in comparison to galantamine, in treatment of human brain
diseases.
[0003] 2. Description of the Related Art
[0004] The diffusion of compounds from the blood plasma into the
brain is complicated by the presence of the blood-brain barrier
that is a membrane that segregates the brain interstitial fluid
from the circulating blood. In designing drugs active in the
central nervous system and able to cross the blood-brain barrier,
one can exploit endogenous active mechanisms, utilize proper
delivery techniques or modify the chemical structure through the
synthesis of pro-drug derivatives.
[0005] Galantamine is an alkaloid that can be isolated from the
bulbs of various snowdrop (Galanthus) and narcissus species
(daffodils, Amaryllidaceae), and recently in particularly high
concentrations from Lycoris radiata, and related species. Synthetic
Galantamine hydrobromide is manufactured by, among other companies,
Sanochemia and Janssen Pharmaceutica. The drug has been approved in
more than 70 nations for the treatment of mild-to-moderate
Alzheimer's disease (AD), a neurodegenerative brain disease.
Extensive studies of the pharmacokinetic profile, tissue
distribution and accumulation of Galantamine in mice, rats, rabbits
and dogs have shown that Galantamine given orally is by no means
preferentially distributed to the brain where it is supposed to
exert its therapeutic activity in said brain diseases. In contrast,
it is accumulated at much higher concentrations in other body
tissues. In male and female rat tissues the highest concentrations
are observed in kidney (tissue to plasma ratio; T/P.about.10-15),
salivary and adrenal gland (T/P.about.7-14), female rat spleen
(T/P.about.20), lung, liver, heart, skeletal muscle and testes
(T/P.about.2-4). In contrast, the brain to plasma ratio is only
T/P.about.1.5. Similarly, the brain/plasma partition coefficient
Kbrain is significantly lower than most other Korgan of
Galantamine.
[0006] Limited penetration ability of Galantamine through the
blood-brain barrier (BBB) into the central nervous system (CNS) is
indicated also by the compound's log P value of 1.3, log P being
defined as the decadic logarithm of the partition coefficient P
which is the ratio of the concentration of compound in aqueous
phase to the concentration of compound in immiscible solvent, as
the neutral molecule. The log P value is obtained by predictive
computational methods and provides a general guideline as to
whether a drug gains rapid access to the CNS, or not. Thus, it has
been established over the past more than 30 years that, assuming
passive absorption, drugs with optimum CNS penetration generally
have log P values around or somewhat above 2. Significantly lower
log P values are often associated with low brain-to-plasma and high
non-brain tissue-to-plasma ratios (see above: log P and T/P ratios
for Galantamine). However, much higher log P values are also of
disadvantage, as high lipophilicity is often associated with
toxicity, non-specific binding, insufficient oral absorption and
limited bioavailability. It follows from this account that BBB
penetration and T/P ratios are essential parameters to be
considered in the case of drugs that are supposed to act mainly or
exclusively in the central nervous system.
[0007] Other important parameters controlling BBB penetration of a
compound are the total polar surface area, the existence of
ionizable groups on the molecule and the affinity of binding to
biological membranes as compared to the affinity of binding to
serum albumin. The latter data set is often used to scrutinize
calculated log P values. In those cases in which special transport
systems do not play a major role for the transport of a compound
through the BBB, the predictions of lipophilicity and BBB
penetration properties are quite suitable for the design of
derivatives that transfer the BBB more efficiently than the parent
compound.
[0008] The present disclosure relates to methods by which the
lipophilicity and/or BBB penetration and/or brain-to-plasma ratio
of a compound is enhanced by formation of a reversible linkage with
one or more suitable groups so as to yield "pro-drugs", i.e.,
chemical derivatives that, after having passed through the
blood-brain barrier, are converted (back) to the original compound
itself inside the patients brain. Liberation of the parent compound
may be by chemical hydrolysis or enzymatic attack, or by redox
reactions. In another embodiment, the present invention refers to
compounds that after chemical modification of the base compound
have achieved a lopP value more favourable for BBB penetration,
with these derivatives acting as such at their target molecules in
the patient's brain.
[0009] The plant alkaloid galantamine has been described as a
cholinesterase inhibitor (ChE-I) and as a nicotinic acetylcholine
receptor (nAChR) sensitizing agent (APL; allosterically
potentiating ligand), and galantamine has been proposed for the
treatment of several human brain diseases, including Alzheimer's
disease (AD). Presently, the compliance of Alzheimer patients to
treatment with ChE-I and APL is rather low, of the order of 20%, a
key reason being the adverse effects nausea, diarrhea, vomiting,
anorexia and muscle cramps. In the case of galantamine, the
majority of these adverse effects is due to actions of the drug
while passing through the gastro-intestinal tract, and to its
rather limited permeation through the blood-brain barrier (BBB)
into the brain. To help patients coping with the adverse effects of
galantamine, the manufacturer's recommended daily dose of the drug
is limited to 16-24 mg per day, and this dose is slowly reached by
stepwise dose increase, beginning at 4 mg/day and over a period of
2-3 months.
[0010] The rather low levels of accumulation of galantamine in the
brain, when administered as the unmodified drug, are a serious
disadvantage with respect to the drug's therapeutic use, i.e., for
the treatment of cognitive disorders, such as AD. As indicated by
the brain-to-plasma ratio of .about.1.3, only a small part of the
administered drug reaches the brain, and the high levels of the
drug in other (peripheral) tissues cause most, if not all, of the
observed adverse effects. The mostly peripheral action of
galantamine is also indicated in its previous use for the treatment
of a number of neuromuscular disorders, including Myasthenia gravis
and poliomyelitis.
[0011] In WO2007/039138 reference is made to the low hydrophobicity
and related limited partition into the human brain of galantamine,
and several procedures for overcoming these drawbacks of a
medication that is supposed to act on target molecules located in
the brain's central nervous system are proposed. In the same
document numerous derivatives of galantamine that significantly
improve transport of the respective compound through the
blood-brain barrier (BBB) are described and they are proposed as
drugs for the treatment of a variety of diseases associated with
cognitive deficits.
[0012] Presently approved drugs for the treatment of Alzheimer's
disease (AD) have in common that they all target excitatory
neurotransmission in the brain, namely the cholinergic and the
glutamatergic systems. Three of the four presently available drugs
(Donepezil, Rivastigmin, Galantamine, Memantine) are cholinergic
enhancers (Donepezil, Rivastigmin, Galantamine) in that they all
inhibit the family of acetylcholine-degrading enzymes denoted as
cholinesterases (ChE). Inhibition of ChE increases the synaptic
concentrations of acetylcholine (ACh), thereby enhancing and
prolonging the action of ACh on muscarinic (mAChR) and nicotinic
(nAChR) acetylcholine receptors. In addition to acting as ChE
inhibitor, Galantamine also acts by allosterically stimulating
(sensitizing) cholinergic receptors. Allosteric sensitization of
nicotinic receptors enhances their activation by ACh or choline
(Ch), thereby correcting for a disease-associated deficit in
transmitter or receptor concentration (Maelicke A & Albuquerque
E X (1996) Drug Discovery Today 1, 53-59; Maelicke A &
Albuquerque E X (2000) Eur J Pharmacol 393, 165-170). In addition
to their therapeutic benefits, these drugs induce adverse
peripheral and central side effects; the muscarinic ones including
nausea, vomiting and diarrhea, and the nicotinic ones including
tremors and muscle cramps. From meta data (Cochrane reviews,
(2004), Issue 4) and direct comparison clinical studies (Wilcock G
K et al. (2000) Brit Med Journ 321:1-7), the relatively weakest of
the three presently used ChE inhibitors, Galantamine, has the
highest clinical efficacy, with the therapeutic benefit achieved at
concentrations that are well below those required for effective
inhibition of AChE (Raskind M A et al. (2000) Neurology 54,
2261-2268; Maelicke A & Albuquerque E X (2000) Eur J Pharmacol
393, 165-170). It has been suggested that the higher therapeutic
efficacy of Galantamine, as compared to the other two available ChE
inhibitors, is due to an additional or alternative mode of action,
i.e., allosteric sensitization of nAChR (Maelicke A &
Albuquerque E X (1996) Drug Discovery Today 1, 53-59).
[0013] Galantamine enhances nicotinic cholinergic neurotransmission
by acting directly on nicotinic receptors (Schrattenholz A et al.
(1996) Mol Pharmacol 49, 1-6; Samochocki M et al. (2003) J
Pharmacol Exp Therap 305, 1024-1036). The drug binds to a distinct
allosteric site on these receptors (Schroder B et al. (1993) J Biol
Chem 269, 10407-10416), from which it acts synergistically with
acetylcholine (or choline) to facilitate nAChR activation (Maelicke
A & Albuquerque E X (1996) Drug Discovery Today 1, 53-59;
Maelicke A & Albuquerque E X (2000) Eur J Pharmacol 393,
165-170). Compounds acting like Galantamine in this way are
referred to as "allostericaly potentiating ligands (APL)"
(Schrattenholz A et al. (1996) Mol Pharmacol 49, 1-6, Maelicke A
& Albuquerque E X (2000) Eur J Pharmacol 393, 165-170).
[0014] The APL action on human nicotinic receptors has been
demonstrated by electrophysiological studies using human brain
slices (Alkondon, M. et al., (2000) J Neurosci 20, 66-75) and human
recombinant cell lines each expressing a single nAChR subtype
(Samochocki M et al (2000) Acta Neuro Scand Suppl 176, 68-73,
Samochocki M et al. (2003) J Pharmacol Exp Therap 305, 1024-1036).
All human nAChR subtypes analysed so far are sensitive to
enhancement by APL. In the presence of Galantamine, the binding
affinity and channel opening probability of nAChR are increased,
leading to a decrease in EC50 for ACh between 30% and 65%
(Samochocki M et al (2000) Acta Neuro Scand Suppl 176, 68-73,
Samochocki M et al. (2003) J Pharmacol Exp Therap 305, 1024-1036).
Furthermore, Galantamine increases the slope of the dose-response
curve for ACh, which has been interpreted as an increase in the
cooperativity between nAChR subunits (Maelicke A & Albuquerque
E X (1996) Drug Discovery Today 1, 53-59).
[0015] The APL effect of Galantamine is observed at submicromolar
concentrations (Samochocki M et al (2000) Acta Neuro Scand Suppl
176, 68-73, Samochocki M et al. (2003) J Pharmacol Exp Therap 305,
1024-1036), i.e., below the concentration range at which ChE
inhibition takes place. The two modes of action of nicotinic APL
are independent of each other, as was shown by ion flux studies
(Okonjo K et al (1991) Eur J Biochem 200, 671-677; Kuhlmann J et al
(1991) FEBS Lett 279, 216-218) and electrophysiological studies of
brain slices from both rats and humans (Santos M D et al (2002) Mol
Pharmacol 61, 1222-1234). In these studies, when cholinesterase
activity was completely blocked by either reversible or
irreversible blocking agents, the nicotinic APL, e.g., Galantamine,
still was able to produce an APL effect of the same size as in the
absence of the other ChE inhibitors. Of the cholinesterase
inhibitors presently approved as AD drugs, Galantamine is the only
one with nicotinic APL activity (Maelicke A et al (2000) Behav
Brain Res 113, 199-206).
[0016] The use of Galantamine and other APL as a drug treatment
strategy for cognitive disorders, including AD and PD was proposed
in 1996 (Maelicke A & Albuquerque E X (1996) Drug Discovery
Today 1, 53-59). Later, the proposal was extended to vascular and
mixed dementia (Maelicke A et al (2001) Biol Psychiatry 49,
279-288), schizophrenia, epilepsy and other diseases with a
nicotinic cholinergic deficit.
[0017] The comparatively low levels of accumulation of Galantamine
in the brain are a serious disadvantage with respect to the drug's
therapeutic use, i.e., for the treatment of cognitive disorders,
such as AD. As indicated by the T/P ratios, only a small part of
the administered drug reaches the brain, and the high levels of the
drug in other (peripheral) tissues may be responsible for some of
the observed adverse side effects. As a point in case, long before
having been approved for the treatment of AD, Galantamine has
primarily been used for the treatment of a number of neuromuscular
disorders, including Myasthenia gravis and poliomyelitis.
[0018] EP-A 648 771, EP-A 649 846 and EP-A 653 427 all describe
Galantamine derivatives, a process for their preparation and their
use as medicaments, however none of these applications considers
ways and means of enhancing penetration through the blood-brain
barrier and brain-to-plasma ratio of base compounds and
derivatives.
[0019] U.S. Pat. No. 6,150,354 refers to several Galantamine
analogues for the treatment of Alzheimer's disease. However,
selective chemical modification for the purpose of increasing
penetration through the blood-brain barrier is not considered.
[0020] WO 01/74820, WO 00/32199 and WO 2005030333 refer to
derivatives and analogues of Galantamine for the treatment of a
variety of human brain and other diseases, and acute functional
brain damage. However, selective chemical modifications or other
means of improving blood-brain barrier penetration are not
considered.
[0021] WO 88/08708, WO 99/21561, WO 01/43697 and US 2003/0162770
refer to derivatives and analogues of Galantamine for the treatment
of various cognitive symptoms. However, selective chemical
modifications or other means of improving blood-brain barrier
penetration are not considered.
[0022] WO 2005/030713 refers to a method for the synthesis of
optical isomers of Galantamine from a Narwedine bromoamide
derivative. However, it does not deal with other derivatives of
Galantamine, or their use as medicaments, or chemical modifications
aimed at enhancing blood-barrier penetration of said compounds.
[0023] WO 97/40049 describes several derivatives of benzazepines
and related compounds that may be applied for the treatment of
Alzheimer's disease. However, no concept is provided in this
application for increasing the penetration of compounds through the
blood-brain barrier.
SUMMARY OF THE INVENTION
[0024] Some embodiments provide compounds usable as pro-drugs or as
a medicament having high pharmacodynamic effects in the brain's
central nervous system and low peripheral side effects.
[0025] Compounds disclosed herein include those described by
formula (III):
##STR00001##
wherein the bond <1> to >2> is a single or a double
bond and the bond between <3> and R1 is a single or a double
bond and bond <10> to <11> is a single or no bond and
residues are [0026] R1: OH, OCO-(3-pyridyl)(=nicotinic acid
residue), OCO-(3-methyl-3-pyridyl), OCO--(C.sub.1-C.sub.6 alkyl),
OCO--(C.sub.1-C.sub.21 alkenyl), OCO--NH--(C.sub.1-C.sub.6 alkyl),
OCO--(CH.sub.2).sub.x--NH--COO--(C.sub.1-C.sub.6 alkyl),
O--CH.sub.2--O--(C.sub.1-C.sub.6 alkyl),
O--(CH.sub.2).sub.x--OCO--(C.sub.1-C.sub.6 alkyl),
O--(CH.sub.2).sub.x--OCO--(CH.sub.2).sub.x--N--COO--(C.sub.1-C.sub.6
alkyl), O--(CH.sub.2).sub.x--OCO--(CH.sub.2).sub.y-aryl,
OCOO--(C.sub.1-C.sub.6 aminalkyl),
OCOO--(CH.sub.2).sub.x-tetrahydrofuranyl, or a sugar, preferably
glucuronic acid residue, wherein x=1, 2, 3 or 4 and y=0, 1, 2, 3 or
4; [0027] wherein if bond <3> to R1 is a double bond, then
R1=O, NH, NOH, NOR6, N--CO--NH.sub.2, N--CS--NH.sub.2,
N--C(.dbd.NH)--NH.sub.2, N--NH-phenyl, N--NHR6, N--N(R6).sub.2,
N--N.dbd.(CH.sub.2).sub.n, with R6=C.sub.1-C.sub.5 unbranched or
branched, saturated or unsaturated (ar)alkyl, phenyl or benzyl and
n=2-8; and [0028] wherein if bond <3> to R1 is a single bond,
then R1=OH, SH, NH.sub.2, NHR6, N(R6).sub.2, OR7, O--CR8R9,
O--CO--CHR10, NR11R12, or O--CO--R14, [0029] with
R7=C.sub.1-C.sub.22 unbranched or branched, (poly-)unsaturated or
saturated alkyl, optionally containing an additional (ar)alkoxy or
di(ar)alkylamino group, a sugar or sugar derivative residue,
preferably glucuronic acid residue, a phosphoryl, alkylphosphoryl
or arylphosphoryl group, a sulfatyl or alkylsufatyl group, or
COR13, [0030] where R13=R6 or R7 or pyridyl or dihydropyridyl or
OR6, preferably methyl, 3-pyridyl, 4-pyridyl, 3-dihydropyridyl,
4-dihydropyridyl [0031] R8 and R9 are the same or different and any
of H, Me, Ph or they together form a spiro-ring --(CH.sub.2)n- with
n=4-6 [0032] R10=H or the side chain of a natural amino acid
including R10, R11 together are forming a proline or
hydroxy-proline derivative [0033] R11 either is together with R10
forming a proline or hydroxy-proline derivative or is H [0034] R12
is a carbamate protecting group including t-butoxycarbonyl,
benzyloxycarbonyl and other N-protecting groups; [0035] R14 is an
aromatic or heteroaromatic 5- or 6-membered ring, selected from
substituted benzene with the proviso that it is not 2-fluorobenzene
or 3-nitro-4-fluorobenzene, optionally substituted naphthaline,
thiophene, pyrrole, imidazole, pyrazole, oxazole, thiazole; or
CH(C.sub.2H.sub.5)CH.sub.3, CH.sub.2--C(CH.sub.3).sub.3, or
cyclopropane; [0036] R2: R7, or O--CR8R9, O--CO--CHR10, or NR11R12
with the same definitions of R7-R12 as above, H, CH.sub.3,
CO--(C.sub.1-C.sub.6 alkyl), CH.sub.2--OCO--(CH.sub.2).sub.x-aryl,
or a sugar, preferably glucuronic acid residue; [0037] R3: H, F,
Cl, Br, I, NH.sub.2, NO.sub.2, CN, CH.sub.3; [0038] R4: H,
C.sub.1-C.sub.6 alkyl, preferably CH.sub.3, CO--(C.sub.1-C.sub.6
alkyl), CO-(3-pyridyl)(=nicotinic acid residue),
CO-(3-methyl-3-pyridyl),
CO--(CH-mercaptoalkyl)-(CH.sub.2).sub.x-aryl,
(CH.sub.2).sub.x--OCO--(CH.sub.2).sub.x--N--COO--(C.sub.1-C.sub.6
alkyl),
(CH.sub.2).sub.x--OCO--(CH-arylalkyl)-N--COO--(C.sub.1-C.sub.6
alkyl), wherein x=1, 2, 3 or 4; [0039] R5: if R4=H, then R5 is an
electron pair; [0040] if R4=CH.sub.3 then R5 is an electron pair,
hydrogen or a C.sub.1-C.sub.5 (ar)alkyl group,
CH.sub.2--O--CH.sub.3, CH.sub.2--O--CO--R6, CH.sub.2--O--CR8R9,
O--CO--CHR10, or NR11R12 with the same definitions of R6 and R8-R12
as above, and wherein the nitrogen at position <10> has an
additional positive charge as well as a counterion, selected from
chloride, bromide, iodide, sulphate, nitrate, hydrogensulfate,
phosphate, methanesulphonate, tosylate or any other
pharmaceutically acceptable anion, with the proviso that the
resulting compound is not Galantamine, Norgalantamine, Sanguinine,
Norsanguinine, Lycoramine, Norlycoramine, Lycoraminone, Narwedine,
Nornarwedine, 3-Amino-3-deoxy-galantamine or
3-amino-3-deoxy-1,2-dihydro-galantamine; [0041] or
R5=(CH.sub.2).sub.x--O--(C.sub.1-C.sub.6 alkyl),
(CH.sub.2).sub.x--OCO--(C.sub.1-C.sub.6 alkyl),
(CH.sub.2).sub.x--OCO--(CH.sub.2).sub.x-aryl,
(CH.sub.2).sub.x--OCO--(CH.sub.2).sub.x--N--COO--(C.sub.1-C.sub.6
alkyl), wherein x=1, 2, 3 or 4; wherein when bond <10> to
<11> is a single bond the nitrogen at position <10> has
a positive charge and the counterion is chloride, with the proviso
that the compound is not Galantamine, Norgalantamine, Sanguinine,
Norsanguinine, Lycoramine, Norlycoramine, Lycoraminone, Narwedine,
Nornarwedine, 3-Amino-3-deoxy-galantamine or
3-amino-3-deoxy-1,2-dihydro-galantamine as a pro-drug or medicament
with improved blood-brain barrier permeability compared to
Galantamine.
[0042] Other embodiments relate to procedures for achieving a
favorable distribution ratio of brain to periphery for antidementia
drugs of various kinds, including cholinergic receptor sensitizing
agents, cholinesterase inhibitors and neuroprotective drugs.
[0043] In this way the therapeutic effect-to-dose ratio can be
increased and adverse side-effects can be reduced when the drugs
are administered as medicaments for the diseases mentioned in the
present application. This object is particularly met e.g., by
site-specific chemical modification (derivatization) of said
compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1: 125 chemical structures and log P values of new
compounds that (i) act as cholinergic enhancers, and/or (ii) have
higher log P-values than Galantamine (Galantamine included in Table
4 for comparison).
[0045] FIG. 2: Brain esterase inhibition by galantamine and several
pro-galantamines. A 20% mouse brain homogenate was used,
supplemented with 200 .mu.M of acetylthiocholine as substrate, and
the initial reaction kinetics were measured according to Riddles P
W, Blakeley R L, Zerner B., "Reassessment of Ellman's reagent"
Methods Enzymol. 1983, 91:49-60. As shown in the figure, even 50
.mu.M of the respective pro-galantamines were unable to achieve a
level of inhibition of brain cholinesterase that is comparable in
size to that of 1 .mu.M galantamine. A non-cleavable galantamine
derivative (Gln 1063) leads to negative values. In derivative Gln
1063 R1 in Formula V is
--O--Si(CH.sub.3).sub.2--C(CH.sub.3).sub.2--C(CH.sub.3).sub.2H.
[0046] FIG. 3: Enzymatic cleavage of pro-galantamine to
galantamine. Butyrylcholinesterase, 25 units/ml, was used. Reaction
temperature was 37.degree. C. Appearance of the fluorescent
reaction product galantamine was determined by fluorescence
detection.
[0047] FIG. 4: Interaction of galantamine and pro-galantamine with
a4.beta.2 neuronal nicotinic acetylcholine receptor ectopically
expressed in HEK-293 cells. The increase in response to
acetylcholine in the presence of galantamine and Gln-1062,
respectively, was determined by whole-cell patch clamp recording.
Galantamine achieved a maximal enhancement of response of
.about.40% whereas the pro-galantamine achieved a maximal
enhancement of only .about.17%.
[0048] FIG. 5: Pharmacokinetics of pro-galantamine Gln-1062 (3
mg/kg) in the mouse. 5a shows the measurable concentration of
applied pro-galantamine and the resulting concentration of
galantamine by cleavage of the pro-galantamine in brain and blood.
The curve starting with the highest concentration refers to
derivative GLN-1062, which is benzoyl-galantamine, in brain. FIG.
5b is an excerpt ("zoom") of FIG. 5a, showing the concentration
range between 0.00 and 1.00 .mu.g/g (substance/body weight) more in
detail. In FIG. 5b the curve above refers to the concentration of
resulting galantamine in brain, the middle curve shows the
concentration of galantamine in blood and the curve starting with a
concentration of about 0.3 .mu.g/g and decreasing refers to the
concentration of GLN-1062 in blood.
[0049] FIG. 6: Behavioral index for gastro-intestinal side effects
in ferrets following application of galantamine and several
R1-pro-galantamines, respectively.
[0050] FIG. 7: Reversal from scopolamine-induced amnesia in mice,
in the presence of galantamine and several R1-pro-galantamines,
respectively. Scopolamine induces a memory deficit that can be
measured by increased alternation in a T-maze trial. The cognition
enhancing drugs were i.p. injected at several different doses
together with scopolamine 20 min before a T-maze trial. Recovery
was measured as a function of dose, and the EC.sub.50 was
determined for each drug.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0051] The present disclosure relates to significant enhancement in
the brain-to-plasma ratio of cholinergic receptor sensitizing
agents, such as the APL Galantamine (and related compounds), which
is achieved by administering not the drug itself but a "pro-drug"
that is converted (back) to the drug itself inside the brain of the
patient. As another means for improving penetration through the
blood-brain barrier (BBB) and thereby the therapeutic efficacy of
the drug, the compounds themselves have been chemically modified so
as to not only having larger efficacy as nicotinic APL and/or as
neuroprotective agent, but in addition having enhanced
lipophilicity (higher log P) or otherwise improved BBB transport
properties. Due to these improvements, the pro-drugs and other
compounds addressed in this application should be significantly
more potent as medicaments for the treatment of cognitive disorders
than is, for example, Galantamine. The invention applies to the
compounds, selected pro-drugs and pharmaceutically acceptable salts
thereof, which might be administrated via the mouth, blood, skin,
by nasal application, or any other suitable application route.
[0052] Herein the term "pro-drug" refers to a derivative of a base
compound wherein the group(s) added or replaced on said base
compound are cleaved or returned to the group originally contained
in the base compound when the derivative has reached the area or
site of action. Thus, in case of a "pro-drug", an effective agent
is administrated as a derivative (which is said pro-drug), however,
the compound mainly or exclusively effective at the target site
within the brain is the agent itself, not the derivatized compound
or metabolites thereof.
[0053] The term "derivative" refers to any change of a base
compound defined in the present application. The term "derivative"
is used to describe a compound which either can be a pro-drug, or
can be an effective agent itself/in its own right or in the
derivatized form.
[0054] The terms "sensitizing agent" and "allosterically
potentiating ligand, APL" refer to effectors that enhance
cholinergic neurotransmission by direct interaction via an
allosteric site with cholinergic receptors.
[0055] The terms "cholinergic enhancer" and "cholinergic agent"
refer to compounds that enhance/modulate cholinergic
neurotransmission by inhibition of cholinesterases, by allosteric
sensitization and/or direct activation of cholinergic receptors
and/or by activating/modulating relevant intracellular pathways via
second messenger cascades.
[0056] A derivative or pro-drug has an "enhanced blood-brain
barrier permeability" according to the present invention or an
"enhanced blood-brain barrier penetration" if, after administration
of a pro-drug or derivative thereof to a living organism, a higher
amount of said compound penetrates through the BBB, resulting in a
higher level of effective agent in the brain, as compared to
administration of the base compound without derivatization. The
enhanced BBB penetration should result in an increased
brain-to-tissue ratio of the effective agent compared to the ratio
of the base compound. Methods for determination of an enhanced BBB
permeability are disclosed in this application (see supra).
[0057] The "base compound" according to the present invention
preferably is Galantamine, Norgalantamine, Narwedine,
N-Demethylnarwedine, Lycoramine, Lycoraminone, Sanguinine,
Norsanguinine, and others (see table 1).
[0058] "log P" is defined as the decadic logarithm of the partition
coefficient P which is the ratio of the concentration of a compound
in aqueous phase to the concentration of a compound in immiscible
solvent, as the neutral molecule.
[0059] The term "alkyl" shall mean a straight, branched or cyclic
alkyl group of the stated number of carbon atoms. Examples include,
but are not limited to methyl, ethyl, n-propyl, iso-propyl,
n-butyl, isobutyl, sec-butyl, t-butyl, and straight and branched
chain pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,
pentadecyl etc. . . . or the according cyclic alkyls.
[0060] The term "halo" shall mean chloro, fluoro, bromo and
iodo.
[0061] The term "aryl" shall mean phenyl having 0, 1, 2 or 3
substituents independently selected from the group of alkyl,
alkoxy, alkylcarbonyl, halo- or trihalomethyl.
[0062] The term "cycloalkyl" shall mean a cycloalkyl group of from
3 to 12 carbon atoms and including multiple ring alkyls such as for
example, adamantyl, camphoryl, and 3-noradamantyl.
[0063] In any case when a range between two limits is described it
is meant that any value or integer in this range is disclosed. For
example "C.sub.1-C.sub.8" means C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7 or C.sub.g; or "between 0.1 and 1" means
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.
[0064] A "natural amino acid" is any amino acid naturally occurring
in biochemical pathways or in peptides/proteins. These are
particularly alanine, asparagine, cysteine, glutamine,
phenylalanine, glycine, histidine, isoleucine, methionine, proline,
glutamate, arginine, serine, threonine, valine, thryptophane,
tyrosine, their methylated forms or the according salts.
[0065] With "sugar" is meant any suitable sugar, either an aldose
or ketose, a pyranose or furanose, heptose or hexose, mono- or
polysaccharide, like e.g., glucuronic acid, glucose, fructose,
galactose, mannose, saccharose, lactose, maltose etc., however,
glucuronic acid is preferred.
[0066] The main focus of the present invention is to improve
blood-brain barrier permeability, by increasing the lipophilicity
or the transport properties, or the ability of passing the
blood-brain barrier, of compounds that are known to act as
effective agents in correcting a cholinergic deficit, e.g., APL of
nicotinic receptors or inhibitors of cholinesterases.
[0067] In one preferred embodiment the present invention refers to
a method for increasing blood-brain barrier penetration of a
cholinergic enhancer by preparing derivatives (either formally by
their chemical structure or directly by chemical synthesis) of a
molecule with a base structure of the general formula (I):
##STR00002##
[0068] wherein the bond between positions <1> and <2>
as well as <11> and <12> denotes a single- or double
bond, and the bond between <10> and <11> is either a
single bond or no bond. [0069] R1=.dbd.O, .dbd.NOH,
.dbd.NH--NHCH.sub.3, --OH, --OCOCH.sub.3, --NH.sub.2, or a
(substituted) derivative of the ketone, like semicarbazone,
thiosemicarbazone, aminoguanidine etc. [0070] R2=H, CH.sub.3,
acetyl [0071] R3=H, CH.sub.3, F, Cl, Br, I [0072] R4=H,
CH.sub.3.
[0073] In Table 1, compounds are exemplified with a base structure
of the general formula (II)
##STR00003##
that belong to the structures summarized in formula (I):
TABLE-US-00001 TABLE 1 Bond Bond logP R1 R2 R3 R4
<1>-<2> <3>-R1 Name calcd.(1) OH CH.sub.3 H
CH.sub.3 Double Single Galantamine 1.30 OH CH.sub.3 H H Double
Single Norgalantamine 1.38 OH H H CH.sub.3 Double Single Sanguinine
0.83 OH H H H Double Single Norsanguinine 0.91 MeCH(OH) H H
CH.sub.3 Double Single Leucotamine 1.23 CH.sub.2--CO OH CH.sub.3 H
CH.sub.3 Single Single Lycoramine 1.28 OH CH.sub.3 H H Single
Single Norlycoramine 1.36 O CH.sub.3 H CH.sub.3 Single Double
Lycoraminone 0.85 O CH.sub.3 H CH.sub.3 Double Double Narwedine
0.74 O CH.sub.3 H H Double Double Nornarwedine 0.82 NH2 CH.sub.3 H
CH.sub.3 Double Single 3-Amino-3-deoxy- 1.05 galantamine NH2
CH.sub.3 H CH.sub.3 Single Single 3-amino-3-deoxy-1,2- 0.89
dihydro-galantamine (1)Calculated using Advanced Pharma Algorithms
Software ToxBoxes V1.0.2
[0074] The compounds listed in Table 1, and other compounds to be
used as a base compound for derivatization according to the present
invention, can be obtained either by isolation from natural sources
or by total chemical synthesis, or by chemical modification of
natural or synthetic compounds.
[0075] The compounds to be used according to the present invention
can be derivatives of the above listed molecules that can be
demonstrated to act as cholinergic enhancers. This property of said
derivatives may be manifested by one or more of the following
properties; by their ability to sensitize cholinergic receptors,
and/or inhibit brain cholinesterases, and/or modulate intracellular
messenger levels, and/or act neuroprotective. The ability to act as
sensitizing agent on nicotinic receptors can be determined by
electrophysiological and Ca-imaging methods, as described in
Schrattenholz A et al. (1996) Mol Pharmacol 49, 1-6 and Samochocki
M et al (2000) Acta Neuro Scand Suppl 176, 68-73; Samochocki M et
al. (2003) J Pharmacol Exp Therap 305, 1024-1036. The ability to
inhibit cholinesterases can be determined by the photometric method
of Ellman et al., Biochem. Pharmacol. 7, 88 (1961). The ability to
modulate intracellular messenger levels can be determined by
Ca-imaging methods (Samochocki M et al. (2003) J Pharmacol Exp
Therap 305, 1024-1036) and other means of recording changes in
intracellular messenger levels or effects resulting thereof (Kihara
T et al (2004) Biochem Biophys Res Commun 325, 976-982). The
ability to act neuroprotective can be determined by a variety of in
vitro and in vivo test systems, including in cell culture (Arias E
et al (2003) Neuropharmacol 46, 103-1S 14; Kihara T et al (2004)
Biochem Biophys Res Commun 325, 976-982) and in animal models of
neurodegenerative diseases (Capsoni et al (2002) Proc Natl Acad Sci
USA 99, 12432-12437).
[0076] As specific examples, Table 2 exemplifies compounds that are
derivatives of a base structure of the following general formula
(III)
##STR00004##
and act in any way as cholinergic enhancers:
TABLE-US-00002 TABLE 2 Bond Bond Bond Bond logP R1 R2 R3 R4 R5 1-2
3-R1 10-11 11-12 Name calcd (1) OH CH.sub.3 H CH.sub.3 CH.sub.3 D S
n S 10,11-Seco- 2.67 10-methyl- galantamine OH CH.sub.3 H CH.sub.3
H D S n D 10,11-Seco- 2.09 11,12- dehydro- galantamine NOH CH.sub.3
H CH.sub.3 e D D S S Narwedinoxim 1.15 NNHCH.sub.3 CH.sub.3 H
CH.sub.3 e D D S S Narwedin-N- 0.34 methyl- hydrazone OH CH.sub.3 F
CH.sub.3 e D S S S 8-Fluoro- 1.25 galantamine OH CH.sub.3 Br
CH.sub.3 e D S S S 8-Bromo- 2.27 galantamine OH CH.sub.3 I CH.sub.3
e D S S S 8-Iodo- 2.26 galantamine OH CH.sub.3 Br CH.sub.3 O D S S
S 8-Bromo- 2.68 galantamine- N-oxide OH H H CH3 E D S S S
Sanguinine 0.83 O CH.sub.3 H CH.sub.3 e D D S S Narwedin 0.74 O
CH.sub.3 CH.sub.3 CH.sub.3 e D D S S 8-Methyl- 1.15 narwedine
O--CO--(CH.sub.2)11--CH.sub.3 CH.sub.3 H CH.sub.3 e D S S S
GLN-0962 7.42 O--CO--(CH2)6--CO- CH.sub.3 H CH.sub.3 e D S S S
GLN-0971 7.80 gal-6-yl O--CO--(CH.sub.2).sub.11--CH.sub.3
CO--(CH.sub.2).sub.11--CH.sub.3 H CH.sub.3 e D S S S GLN-0935 11.7
(1) Calculated using Advanced Pharma Algorithms Software ToxBoxes
V1.0.2. Abbreviations: s: single bond; d: double bond; n: no bond;
e: electron pair
[0077] Most of the compounds listed in Table 2 are not only
efficacious agents in one or more of the tests cited above, but
most of them also have more favourable log P and/or transport
properties than the base compounds from which they are derived.
[0078] To further improve BBB permeability and brain/plasma
distribution ratio, modifications of the following kinds can be
performed so as to make the compounds exemplified in Tables 1 and 2
more lipophilic or enhance otherwise their transport into the CNS,
in comparison to the base compound: [0079] 1. Conjugations to
groups or molecules that are known to occur in the course of
metabolic conversions, e.g., carbohydrate conjugates such as
glycosyls, glucuronides and natural metabolites, or are otherwise
known to readily pass the blood-brain barrier, e.g., amino acids,
vitamins, various messenger molecules and drugs. [0080] 2.
Conjugations to groups leading to quaternary ammonium salts with a
labile nitrogen-carbon bond (see e.g., Example 1). [0081] 3.
Conjugations to groups leading to esters, e.g., acylderivates with
enhanced lipophilicity and BBB penetration properties. For example,
such compounds may be esters of the oxygen function in position 3
and/or 6 of the following base structure (IV):
[0081] ##STR00005## [0082] a) Esters with saturated or unsaturated
fatty acids containing 1-22 carbon atoms optionally containing an
additional (ar)alkoxy or di(ar)alkylamino group [0083] b) Esters
with carbonic acid where one acidic function of carbonic acid is
esterified with the 3- and/or 6-position of galantamine and the
other represents an ester as defined in 3a. [0084] c) Esters with
(substituted) pyridine- or (substituted) dihydropyridine-carboxylic
acids (see e.g., Example 2) [0085] d) Esters with phosphoric and
sulfonic acids [0086] 4. Formation of ketals or aminals of
substituents in positions 3, 6, and 10 that increase the
lipophilicity and are hydrolyzed to the desired derivatives, e.g.,
(nor)galantamine derivatives (see e.g., Examples 3 and 4) [0087] 5.
Formation of basic and/or quaternary carbamates of said compounds
that are chemically or metabolically unstable. [0088] 6.
Conjugation to a lipophilic dihydropyridinium carrier, e.g., as
1,4-dihydro-1-methyl-3-pyridinecarboxylate, that in the brain is
enzymatically oxidised to the corresponding ionic pyrimidinium
salt. [0089] 7. Conjugation with nicotinic acid, nicotinic acid
amide, various cofactors, messenger molecules and other chemical
entities that enhance lipophilicity and transport through the
BBB.
[0090] These modifications lead to compounds of the following
general formula (III)
##STR00006##
wherein the bond between positions <1> and <2> denotes
a single- or double bond, with the proviso that the structure is
not any of those listed in Table 1 and the bonds <1> to
<2> and <11> to <12> can be either a single or a
double bond, and the bond between <10> and <11> is
either a single bond or no bond and the residues R1-R5 are defined
as follows:
R1:
[0091] a) if bond <3> to R1 is a double bond, then [0092]
R1=O, NH, NOH, NOR6, N--CO--NH.sub.2, N--CS--NH.sub.2,
N--C(.dbd.NH)--NH.sub.2, N--NH-phenyl, N--NHR.sub.6,
N--N(R6).sub.2, N--N.dbd.(CH.sub.2).sub.n [0093] with
R6=C.sub.1-C.sub.5 unbranched or branched, saturated or unsaturated
(ar)alkyl, phenyl or benzyl and n=2-8 [0094] b) if bond <3>
to R1 is a single bond, then [0095] R1=OH, SH, NH.sub.2, NHR6,
N(R6).sub.2, OR7, O--CR8R9, O--CO--CHR10, or NR11R12 [0096] with
R7=C.sub.1-C.sub.22 unbranched or branched, (poly-)unsaturated or
saturated alkyl, optionally containing an additional (ar)alkoxy or
di(ar)alkylamino group, a sugar or sugar derivative residue,
preferably glucuronic acid, a phosphoryl, alkylphosphoryl or
arylphosphoryl group, a sulfatyl or alkylsufatyl group [0097] or
COR13, [0098] where R13=R6 or R7 or pyridyl or dihydropyridyl or
OR6, preferably methyl, 3-pyridyl, 4-pyridyl, 3-dihydropyridyl,
4-dihydropyridyl [0099] R8 and R9 are the same or different and any
of H, Me, Ph or they together form spiro-ring --(CH.sub.2).sub.n--
with n=4-6 [0100] R10=H or the side chain of a natural amino acid
including R10 and R11 together are forming a proline or
hydroxy-proline derivative [0101] R11 either is together with R10
forming a proline or hydroxy-proline derivative or is H [0102] R12
is a carbamate protecting group including t-butoxycarbonyl,
benzyloxycarbonyl and other N-protecting groups
R2:
[0102] [0103] H, R7, or O--CR8R9, O--CO--CHR10, or NR11R12, with
the same definitions of R7-R12 as above
R3:
[0103] [0104] H, F, Cl, Br, I, NH.sub.2, NO.sub.2, CN, CH.sub.3
R4:
[0104] [0105] H or CH.sub.3
R5:
[0105] [0106] If R4=H, then R5 is an electron pair [0107] if
R4=CH.sub.3 then R5 is an electron pair, hydrogen or a
C.sub.1-C.sub.5 (ar)alkyl group, CH.sub.2-.beta.-CH.sub.3,
CH.sub.2--O--CO--R6, CH.sub.2--O--CR8R9, O--CO--CHR10, or NR11R12
with the same definitions of R6 and R8-R12 as above.
[0108] In all the latter cases, the nitrogen bears an additional
positive charge as well as a counterion, selected from chloride,
bromide, iodide, sulphate, nitrate, hydrogensulfate, phosphate,
methanesulphonate, tosylate or other pharmaceutically acceptable
anion.
[0109] Preferred derivatives of the main concept of the invention
are quarternary ammonium salts with a labile nitrogen-carbon bond
at R5; mono- or diacylderivatives (esters) of the hydroxyl groups
of said base compounds (R1, R2); sugar derivatives, preferably
glucuronides (R1, R2); derivatives coupled with nicotinic acid (R1,
R2); and selected halogenides (R3).
[0110] Another preferred derivative of the main concept is a
lipophilic dihydropyridinium carrier. This Redox Chemical Delivery
System (RCDS; Misra A et al (2003) J Pharm Pharmaceut Sci 6,
252-273) is known to significantly enhance drug delivery through
the BBB into the brain parenchyma. Once inside the brain, the
dihydropyridinium moiety is enzymatically oxidized to the
corresponding ionic pyridinium salt. Subsequent cleavage of the
original compound from the carrier leads to liberation of the
original compound and to sustained levels of it in the brain
tissue.
[0111] Other preferred derivatives of the main concept are amino
acids that are known to be transported into the brain by active
amino acid carriers, e.g., tyrosine. Once inside the brain
parenchyma, these derivatives can either directly act on their
target molecules or are first enzymatically liberated before acting
as the original aren't compound.
[0112] As a further aspect of the present invention, the
derivatives obtained by chemical modification do not need to work
as such as medicaments but rather may initially be pro-drugs that,
after penetration though the blood-brain barrier, are converted
(e.g., by brain enzymes) to the parent compound or a metabolite
thereof and work as such as a medicament. Said pro-drug or
derivative is used to prepare a medicament or pharmaceutical
composition that preferably can be used for the treatment of brain
diseases associated with a cholinergic deficit.
[0113] Of the derivatives contained in the general structure of
formula (III) and with the proviso and definitions provided there,
the following are of particular interest in regard to the present
invention, as they have not yet been described or developed under
the premise of having higher lipophilicity and/or better BBB
transport properties and/or higher brain-to-plasma ratio than their
parent compounds (Table 1) from which they are derived by chemical
modification:
TABLE-US-00003 TABLE 3 Examples of compounds described in previous
publications/patents presently shown that they (i) act as
cholinergic enhancers, and/or (ii) have higher logP-values than
Galantamine STRUCTURE logP Name ##STR00007## 1.30 Galantamine
##STR00008## 1.38 Nor- galantamine ##STR00009## 1.68 3-O-Acetyl-
6-O- demethyl- galantamine ##STR00010## 1.72 8-Bromo- narwedine
##STR00011## 1.76 Narcisine ##STR00012## 1.99 ##STR00013## 2.15
##STR00014## 2.27 ##STR00015## 2.35 ##STR00016## 2.69 ##STR00017##
3.07 ##STR00018## 3.27 ##STR00019## 4.09 ##STR00020## 4.90
Han, So Yeop; Mayer, Scott C.; Schweiger, Edwin J.; Davis, Bonnie
M.; Joullie, Madeleine M. Synthesis and biological activity of
galantamine derivatives as acetylcholinesterase (AChE) inhibitors.
Bioorganic & Medicinal Chemistry Letters (1991), 1(11),
579-80.
[0114] The following derivatives covered by the general structure
of formula (III) and with the proviso and definitions provided
there are particularly preferred derivatives of the main concept of
the invention in that they have not yet been mentioned or described
in any other publication or patent.
[0115] Examples of new compounds that (i) act as cholinergic
enhancers, and/or (ii) have higher log P-values than Galantamine
(the latter for comparison only) are shown in FIG. 1.
[0116] The derivatives shown in Table 3 and FIG. 1 may be used to
prepare a medicament or other pharmaceutical composition. Such
medicament or pharmaceutical composition can be used for the
treatment of a disease state associated with a cholinergic
deficit.
[0117] The usefulness of the derivatives, before and/or after
conversion to the parent compound, to act as effective
pharmaceutical agents is manifested by their ability to sensitize
cholinergic receptors, and/or inhibit brain cholinesterases, and/or
modulate intracellular messenger levels, and/or act
neuroprotective. The ability to act as sensitizing agent on
nicotinic receptors can be determined by electrophysiological and
Ca-imaging methods, as described in Schrattenholz A et al. (1996)
Mol Pharmacol 49, 1-6 and Samochocki M et al (2000) Acta Neuro
Scand Suppl 176, 68-73; Samochocki M et al. (2003) J Pharmacol Exp
Therap 305, 1024-1036. The ability to inhibit cholinesterases can
be determined by the photometric method of Ellman et al., Biochem.
Pharmacol. 7, 88 (1961). The ability to modulate intracellular
messenger levels can be determined by Ca-imaging methods
(Samochocki M et al. (2003) J Pharmacol Exp Therap 305, 1024-1036)
and other means of recording changes in intracellular messenger
levels or effects resulting thereof (Kihara T et al (2004) Biochem
Biophys Res Commun 325, 976-982). The ability to act
neuroprotective can be determined by a variety of in vitro and in
vivo test systems, including in cell culture (Arias E et al (2003)
Neuropharmacol 46, 103-1S 14; Kihara T et al (2004) Biochem Biophys
Res Commun 325, 976-982) and in animal models of neurodegenerative
diseases (Capsoni et al (2002) Proc Natl Acad Sci USA 99,
12432-12437).
[0118] This usefulness can also be ascertained by determining the
ability of these compounds (I) to reduce neuronal cell death and
amyloid plaque formation as well as cognitive impairment in animal
models of Alzheimer's disease (Capsoni et al (2002) Proc Natl Acad
Sci USA 99, 12432-12437) and (2) to enhance learning performance in
various animal test systems. In one particular learning paradigm
applied to old and young rabbits (Woodruff-Pak D et al (2001) Proc
Natl Acad Sci USA, 98, 2089-2094), the classical eye blink
conditioning is used to study the effect of cognition-enhancing
drugs on the septohippocampal cholinergic system. An active test
compound of the present invention will reduce the number of trials
required to learn that the air blow applied onto the animal's eye
does not require the animal to close the eye (eye blink) as a
protective measure.
[0119] This usefulness can also be ascertained by determining the
ability of these compounds to restore deficient memory due to a
cholinergic deficit in the Dark Avoidance Assay (DAA). In this
assay mice are tested for their ability to remember an unpleasant
stimulus for a period of e.g., 24 hours. A mouse is placed in a
chamber that contains a dark compartment; a strong incandescent
light drives it to the dark compartment, where an electric shock is
administered through metal plates on the floor. The animal is
removed from the testing apparatus and tested again, 24 hours
later, for the ability to remember the electric shock administered
in the dark compartment.
[0120] If a nicotinic or muscarinic antagonist, i.e., an
anticholinergic drug that causes memory impairment, is administered
before an animal's initial exposure to the test chamber, the animal
tends to re-enter the dark compartment much sooner than in the
absence of the anticholinergic drug when being placed in the test
chamber 24 hours later. This effect of an anticholinergic drug is
blocked by an active test compound, resulting in a greater interval
before re-entry into the dark compartment.
[0121] The test results may be expressed as the percent of a group
of animals in which the effect of the anticholinergic drug is
blocked or reduced, as manifested by an increased time interval
between being placed in the test chamber and re-entering the dark
compartment.
[0122] According to the present intention and approach, the brain
disease that can be treated with the pro-drugs and derivatives
provided herewith can be any psychiatric, neurological and
neurodegenerative disease associated with a cholinergic deficit of
any kind, including a neurodegenerative loss of cholinergic
neurotransmitters and/or receptors, ACh-synthesizing and
metabolizing enzymes, transport proteins and the like. Such
diseases are exemplified by Alzheimer's and Parkinson's disease,
other types of dementia, schizophrenia, epilepsy, stroke,
poliomyelitis, neuritis, myopathy, oxygen and nutrient deficiencies
in the brain after hypoxia, anoxia, asphyxia, cardiac arrest,
chronic fatigue syndrome, various types of poisoning, anesthesia,
particularly neuroleptic anesthesia, spinal cord disorders,
inflammation, particularly central inflammatory disorders,
postoperative delirium and/or subsyndronal postoperative delirium,
neuropathic pain, subsequences of the abuse of alcohol and drugs,
addictive alcohol and nicotine craving, and subsequences of
radiotherapy, and more. The effect of Galantamine or other
cholinesterase inhibitors in treatment of such diseases are
described e.g., in WO2005/74535, WO2005/72713, WO2005/41979,
WO2005/30332, WO2005/27975, US2004/266659 and WO2004/14393.
[0123] All the derivatives described in the general (base)
structure of formula (III) and Tables 2 and 3 and FIG. 1 either
have an effect as a pro-drug, which means that the derivative,
after entering the brain, is "converted back" into an effective
agent, e.g., Galantamine, Narwedine, Lycoramine, or the other said
base compounds, or they are effective (i.e., as cholinergic
enhancers or agents according to the definition) as derivatives
themselves, meaning that they are not necessarily converted or
metabolised before they act as agents at their target molecules,
e.g., cholinergic receptors or cholinesterases. The common feature
of the derivatives of the present application is that they all
penetrate more effectively through the blood-brain barrier than the
base compound, which according to the present invention preferably
is Galantamine and related compounds. As a result of their improved
BBB penetration properties, these compounds should have higher
therapeutic efficacy and lower adverse side effects than e.g.,
Galantamine.
[0124] The compounds of the present invention whether pro-drugs or
otherwise effective agents can be administered as such or as a
pharmaceutically acceptable salt thereof.
[0125] The derivatives of the common formulae as defined above can
be prepared by any known method, however, it is preferred that the
derivatives are prepared with proper use by the methods described
for derivatization of according compounds in EP-A 649 846 with
reference to scheme I and in the examples; EP-A648 771 with
reference to scheme I and in the examples; EP-A 653 427 with
reference to scheme I and in the examples; U.S. Pat. No. 6,150,354,
paragraph "procedures" and examples; or U.S. Pat. No. 6,638,925,
paragraph "experimental section", respectively. A further reference
is WO 01/74820, wherein combinatory and/or parallel synthesis is
disclosed and synthesis of several compounds is described in the
examples. Further the method can be used as described in Gomes, P.
et al., Rui. Centro de Investigacao em Quimica da Universidade do
Porto, Oporto, Port. Synthetic Communications (2003), 33(10),
1683-1693. A skilled person clearly will understand that in any
case an appropriate educt/appropriate educts has/have to be used to
obtain the desired derivatization of the base structure. The
preparation method is not limiting the invention as long as the
compounds presently described are obtained.
[0126] The compounds of the invention preferably are prepared from
the appropriate optical isomer of Galantamine or Narwedine via the
intermediate 6-demethylgalantamine, a known therapeutically
effective compound, or 6-demethylnarwedine, respectively.
[0127] The pro-drugs and derivatives of this invention are selected
by the following tests, which shall be considered as examples not
limiting the invention: [0128] 1. Activity as nicotinic
"allosterically potentiating ligand (APL), preferentially
determined by electrophysiological methods and, Ca-imaging, using
human cell lines that express individual subtypes of human neuronal
nicotinic acetylcholine receptors (nAChR). [0129] In the case of a
compound acting as such: The activation of nAChR by ACh or agonist
is enhanced in the presence of said compound, with the APL activity
being selectively blocked by antibody FK1. [0130] In the case of a
pro-drug: Enhanced activity as a centrally acting APL after the
pro-drug has been converted to the base compound by treatment with
a rat brain or human brain homogenate extract. [0131] Kinetics of
conversion from pro-drug to drug when incubated with a rat or human
brain extract. [0132] 2. Activity as centrally acting
cholinesterase inhibitor, as tested by various in-vitro, cell
culture and in-vivo test systems. [0133] In the case of a pro-drug:
Enhanced cholinesterase inhibition--or the same level of inhibition
at a significantly reduced dose--is observed when the pro-drug is
administered instead of the original base compound. [0134] Kinetics
of conversion from pro-drug to drug when incubated with a rat or
human brain extract. [0135] 3. Neuroprotective activity in acute
toxicity protection tests (organophosphate poisoning of animals,
in-vitro poisoning by AB and/or glutamate) and in animal models of
neurodegeneraton. [0136] In the case of a pro-drug: Enhanced
neuroprotective activity--or the same level of neuroprotection at a
significantly reduced dose--is observed when the pro-drug is
administered instead of the original base compound. [0137] 4.
Accumulation of the derivatives in the brain of mammals as compared
to unmodified Galantamine or other base compound. [0138] 5.
Lipophilicity, as measured by shake-flask (e.g., octanol/buffer),
HPLC-retention and nanobeads absorption methods. [0139] 6.
Bioconversion t.sub.1/2 in the brain as compared to blood
(systemic). [0140] 7. Theoretical/empirical estimates of
distribution and log P values. [0141] 8. Other miscellaneous
tests.
[0142] As one way of estimating improved lipophilicity of the
derivatized compounds, log P-values are provided in some of the
tables. Improved lipophilicity, as characterized by an increased
log P-value, can either be determined experimentally including HPLC
methods or by predictive computational methods. Although such
calculations cannot replace the experiment, the data are strongly
suggestive as to whether a certain modification of the base
compound will result in an improved lipophilicity. Computer
programs that allow such calculations include e.g., ToxBoxes from
Pharma Algorithms, ACD-Lab, Molecule Evaluator from Cidrux, and
others.
[0143] Another means of estimating the readiness of a compound to
transverse the BBB is by experimental comparison of the membrane
affinity of said compound to its binding affinity to serum albumin,
both determined by the NIMBUS Biotechnology assay (Willmann, S. et
al. (2005) J Med Chem, in print).
[0144] Effective quantities of the compounds of the invention may
be administered to a patient by any of various methods, including
orally as in capsules or tablets, via the skin or by nasal
application. The free base final products, while effective by
themselves, may be formulated and administered in the form of a
pharmaceutically acceptable salt, e.g., for purposes of stability,
convenience of crystallization, increased solubility, release
retardation, and the like.
[0145] Since the pro-drugs/compounds of the present invention pass
the blood-brain-barrier easier than the base compounds, there are
two advantageous aspects: first is the fast uptake of the pro-drug
and therefore a fast onset of effect, second is that the dosage of
application can be decreased compared to known medicaments
resulting in lower peripheral side effects with high efficacy of
the compounds at their effect site (brain). Further the pro-drugs
after passage through the blood-brain-barrier are converted in the
base compound which has a lower permeability through the
blood-brain-barrier, thus the effective compound remains in the
brain, resulting in a longer time period of effectiveness.
[0146] As a representative case, the active compounds of the
present invention may be orally administered, for example, with an
inert diluent or with an edible carrier, or they may be enclosed in
gelatine capsules, or they may be compressed into tablets.
Furthermore, the active compounds of the invention may be
incorporated with excipients and used in the form of tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, chewing
gum and the like. Preferred compositions and preparations according
to the present invention are prepared so that an oral dosage unit
form contains between 0.1 and 50 milligrams of active compound.
[0147] Because BBB penetration and brain-to-plasma ratio of the
compounds modified according to this invention are significantly
enhanced, the dosages of administered drug may be dramatically
reduced, as compared to previous applications, clinical studies and
estimates.
Selected Aromatic and Heterocyclic Derivatives of Galantamine as
Pro-Drugs for the Treatment of Human Brain Diseases
[0148] The proposed derivatives were designed as pro-drugs, in the
sense that they are able to effectively pass the blood brain
barrier (BBB) and, after passing the BBB, they are substrates of
endogenous enzymes and, upon enzymatic cleavage, produce
galantamine. As a result of enzymatic cleavage to galantamine of
such pro-galantamines in the brain, a significantly higher local
concentration of galantamine is achieved in the brain than by
administration of the same dose of original galantamine. The
relatively higher drug concentration in the brain achieved by
pro-galantamine administration will then result in higher efficacy
at a given dose, and the better brain-to-peripheral tissues
distribution will result in fewer or less significant side effects
of treatment. These effects are significant improvements of present
treatment regimens because the efficacy as treatment for brain
diseases of unmodified galantamine (and all other ChE-I presently
approved for this purpose) is rather limited, albeit statistically
significant, possibly due to low dosing. Thus, efficacy is usually
reached only after careful (months-long) up-titration of daily
dose, so as to maintain sufficient compliance of patients to the
largely gastro-intestinal side effects associated with ChE-I
treatment.
[0149] According to some embodiments, it was found that careful
selection concerning the type of substituent and the position of
substitution using galantamine as base structure result in highly
efficacious compounds. Such very efficacious compounds having good
blood brain barrier passing properties and being efficiently
cleaved by an esterase after passage through the BBB are obtained
with compounds having the general formula V
##STR00021##
with R1 being a substituent having particular sterical and
hydrophobic properties.
[0150] These embodiments focus on esters of galantamine that, as
such, have little or no activity as ChE-I and APL compared to
galantamine. Therefore, as long as these compounds remain
uncleaved, they do not interact with the usual target molecules of
galantamine and hence are largely inactive in producing therapeutic
and/or side effects. The reduced reactivity of pro-galantamines is
demonstrated by the following results:
[0151] 1. Significantly reduced activity as ChE-I, as compared to
galantamine.
[0152] 2. Reduced activity as nicotinic APL, as compared to
galantamine.
[0153] 3. Reduced gastro-intestinal side effects, as compared to
galantamine.
[0154] All these approaches were investigated and are explained in
the examples and shown in the figures.
[0155] According to the invention it has been discovered that a
particular group of esters of galantamine display unexpectedly high
brain-to-blood concentration ratios (R.sub.BB-proGal>6, as
compared to R.sub.BB-Gal.about.1.3) and in the brain they are
relatively slowly enzymatically cleaved to galantamine. Therefore,
as is discussed below in more detail, these pro-galantamines are
exceptionally well suited for the treatment of human diseases
associated with cholinergic deficits, such as Alzheimer's disease,
Parkinson's disease, Schizophrenia and a variety of other
psychiatric disorders.
[0156] The esters of galantamine to which the present invention
refers to have the following general structure:
##STR00022##
wherein R1 either is CH(C.sub.2H.sub.5)CH.sub.3,
CH.sub.2--C(CH.sub.3).sub.3, or cyclopropane or being an optionally
substituted aromatic or hetero-aromatic 5- or 6-membered ring.
Specifically, such aromatic and hetero-aromatic rings include
benzene, naphthalene, thiophene, pyrrole, imidazole, pyrazole,
oxazole and thiazole, in case that they are used as medicaments or
pro-drugs for the treatment of neurodegenerative or psychiatric or
neurological disease associated with a cholinergic deficit.
[0157] Such a disease preferably is selected from Alzheimer's and
Parkinson's disease, other types of dementia, schizophrenia,
epilepsy, neuritis, various types of poisoning, anesthesia,
particularly neuroleptic anesthesia, autism, spinal cord disorders,
inflammation, particularly central inflammatory disorders,
postoperative delirium and/or subsyndromal postoperative delirium,
neuropathic pain, subsequences of the abuse of alcohol and drugs,
addictive alcohol and nicotine craving, and subsequences of
radiotherapy.
[0158] The above mentioned compounds were not yet described for the
treatment of such diseases. Furthermore, compounds of formula I
having aromatic or hetero-aromatic 5- or 6-membered ring, selected
from substituted benzene with the proviso that it is not
2-fluorobenzene or 3-nitro-4-fluorobenzene, optionally substituted
naphthalene, thiophene, pyrrole, imidazole, pyrazole, oxazole,
thiazole; or CH(C.sub.2H.sub.5)CH.sub.3,
CH.sub.2--C(CH.sub.3).sub.3, or cyclopropane are according to the
knowledge of the inventor not yet described at all.
[0159] In one preferred embodiment the compounds of the present
invention are selected from
##STR00023##
wherein R2-R6 comprising any substituent selected from H, halogen,
optionally substituted C.sub.1-C.sub.3 alkyl or cyclopropyl, OH,
O-alkyl, SH, S-alkyl, NH.sub.2, NH-alkyl, N-dialkyl, optionally
substituted aryl or hetero-aryl, whereby neighbouring substitutents
can cooperate to form an additional ring.
[0160] In another preferred embodiment of the present invention the
compounds are selected from the group consisting of the compounds
as shown in table A, which is attached below.
[0161] Herein the term "pro-drug" refers to a derivative of
galantamine (base compound) wherein the group(s) added or replaced
on said base compound are cleaved or returned to the hydroxyl group
originally contained in the base compound, when the derivative has
reached the area or site of action. Thus, in case of a "pro-drug",
an effective agent is administrated as a derivative (which is said
pro-drug), however, the compound mainly or exclusively effective at
the target site within the brain is the agent itself, not the
derivatized compound or metabolites other than the base compound
thereof.
[0162] The term "derivative" refers to any change of a base
compound defined in the present application. The term "derivative"
is used to describe a compound which either can be a pro-drug, or
can be an effective agent itself/in its own right or in the
derivatized form.
[0163] The term "pro-galantamine" is used for any derivative of
galantamine described herein which can be cleaved by an enzyme
(esterase) resulting in galantamine.
[0164] The terms "sensitizing agent" and "allosterically
potentiating ligand, APL" refer to effectors that enhance
cholinergic neurotransmission by interaction with an allosteric
site at cholinergic receptors.
[0165] The terms "cholinergic enhancer" and "cholinergic agent"
refer to compounds that enhance/modulate cholinergic
neurotransmission by inhibition of cholinesterases, by allosteric
sensitization and/or direct activation of cholinergic receptors
and/or by activating/modulating relevant intracellular pathways via
second messenger cascades.
[0166] A derivative or pro-drug has an "enhanced blood-brain
barrier permeability" according to the present invention or an
"enhanced blood-brain barrier penetration" if, after administration
of a pro-drug or derivative thereof to a living organism, a higher
amount of said compound penetrates through the BBB of that
organism.
[0167] A compound of the present invention provides an increased
"brain-to-blood concentration ratio" or "brain-to-tissue
concentration ratio" resulting in a higher level of effective agent
in the brain, as compared to administration of the base compound
without derivatization. Methods for determination of an enhanced
BBB permeability are disclosed in WO 2007/039138.
[0168] The "base compound" as well as the "effective agent"
according to the present invention is galantamine. The effective
agent is obtained by (local) enzymatic cleavage of the
derivative.
[0169] "log P" is defined as the decadic logarithm of the partition
coefficient P which is the ratio of the concentration of a compound
in aqueous phase to the concentration of a compound in immiscible
solvent, as the neutral molecule.
[0170] The term "alkyl" shall mean a straight, branched or cyclic
alkyl group. As "alkyl" C.sub.1 to C.sub.10 alkyl groups are
preferred, C.sub.2 to C.sub.8 groups are more preferred and C.sub.2
to C.sub.6 groups are most preferred. C.sub.1 to C.sub.10 means
alkyl groups of the stated number of carbon atoms. Examples
include, but are not limited to methyl, ethyl, n-propyl,
iso-propyl, n-butyl, isobutyl, sec-butyl, t-butyl, and straight and
branched chain pentyl, hexyl, heptyl, octyl, nonyl and decyl etc. .
. . or the according cyclic alkyls.
[0171] The term "halo" shall mean chloro, fluoro, bromo and
iodo.
[0172] The term "aryl" shall mean phenyl having 0, 1, 2, 3, 4 or 5
substituents independently selected from the group of alkyl,
alkoxy, alkylcarbonyl, halo- or trihalomethyl.
[0173] The term "cycloalkyl" shall mean a cycloalkyl group of from
3 to 10 carbon atoms and including multiple ring alkyls such as for
example, adamantyl, camphoryl, and 3-noradamantyl.
[0174] In any case when a range between two limits is described it
is meant that any value or integer in this range is disclosed. For
example "C.sub.1-C.sub.10" means C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9 or C.sub.10;
or "between 0.1 and 1" means 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9 or 1.
[0175] The stereo chemistry of the described derivatives are the
same as that of galantamine.
[0176] Benzoyl esters of galantamine were previously described in
WO 9921561 A1 Davis, Bonnie M. for a method of treatment of
disorders of attention with galantamine, lycoramine, and related
compounds, but no syntheses or analytical or other data were
provided for these compounds.
[0177] Substituted benzoyl esters were previously described in
"Synthesis and biological activity of galantamine derivatives as
acetylcholinesterase (AChE) inhibitors" by Han, So Yeop; Mayer,
Scott C.; Schweiger, Edwin J.; Davis, Bonnie M.; Joullie, Madeleine
M. Dep. Chem., Univ. Pennsylvania, Philadelphia, Pa., USA.
Bioorganic & Medicinal Chemistry Letters (1991), 1(11), 579-80.
CODEN: BMCLE8 ISSN: 0960-894X. Journal written in English. CAN
116:83569 AN 1992:83569 CAPLUS. In this document the synthesis of
several ester and carbamate derivatives of galantamine are
described as well as it was suggested that these compounds are
potential therapeutic agents in the treatment of Alzheimer's
disease, based on their properties as AChE inhibitors.
[0178] In contrast to the teaching of these documents, the
galantamine esters of the present invention have little, if any,
activity as acetylcholinesterase inhibitors but rather are
substrates of said enzyme (see above).
[0179] As representatively demonstrated for the benzoyl derivative
in FIG. 2, these esters have little, if any cholinesterase
inhibitory activity but rather are hydrolysed by cholinesterases to
form galantamine and accordingly act as pro-drugs of galantamine.
As soon as galantamine is generated from these compounds, it acts
as ChE-I and APL, as has previously been described. The structures
of the tested derivatives can be seen in Table 4. As a comparative
derivative a non-cleavable galantamine ether is also tested. Such
derivative results in negative values of inhibition. In derivative
Gln 1063 R1 in formula V is
--O-Si(CH.sub.3).sub.2--C(CH.sub.3).sub.2--C(CH.sub.3).sub.2H.
TABLE-US-00004 TABLE 4 Mol Reg. No. Molecular structure
Abbreviation GLN-1062 ##STR00024## Bz--Gal GLN-1081 ##STR00025##
4-Cl--Bz--Gal GLN-1082 ##STR00026## 4-MeO--Bz--Gal GLN-1083
##STR00027## 4-Me--Bz--Gal GLN-1084 ##STR00028## 3,4-Cl2--Bz--Gal
GLN-1085 ##STR00029## 4-tBu--Bz--Gal GLN-1086 ##STR00030##
3-CF3-4-Cl--Bz--Gal GLN-1088 ##STR00031## 4-CF3--Bz--Gal GLN-1089
##STR00032## 2,4-Cl2--Bz--Gal GLN-1090 ##STR00033## 4-NO2--Bz--Gal
GLN-1091 ##STR00034## 3-Cl--Bz--Gal GLN-1092 ##STR00035##
3-CF3--Bz--Gal GLN-1093 ##STR00036## 3-NO2--Bz--Gal GLN-1094
##STR00037## 3,5-Cl2--Bz--Gal GLN-1095 ##STR00038## 3-Me2N--Bz--Gal
GLN-1096 ##STR00039## 3-Me--Bz--Gal GLN-1097 ##STR00040##
2-Cl--Bz--Gal GLN-1098 ##STR00041## 2,4-F2--Bz--Gal GLN-1099
##STR00042## 2,5-Cl2--Bz--Gal GLN-1100 ##STR00043## 4-F--Bz--Gal
GLN-1101 ##STR00044## 4-NMe2--Bz--Gal GLN-1102 ##STR00045##
4-NH2--Bz--Gal GLN-1103 ##STR00046## 3-Me-4-NMe2--Bz--Gal GLN-1104
##STR00047## 3,4-OCH2O--Bz--Gal GLN-1105 ##STR00048## 4-Ac--Bz--Gal
GLN-1113 ##STR00049## 2-AcO--Bz--Gal GLN-0978 ##STR00050##
n-prop--Gal GLN-0979 ##STR00051## i-but--Gal GLN-0992 ##STR00052##
GLN-0993 ##STR00053## n-Hex--Gal GLN-1011 ##STR00054##
neo-pent--Gal GLN-1060 ##STR00055## GLN-1061 ##STR00056## GLN-1067
##STR00057## R/S-i-pent--Gal GLN-1069 ##STR00058## GLN-1070
##STR00059## GLN-1071 ##STR00060## GLN-1076 ##STR00061## CyBu--Gal
GLN-1077 ##STR00062## GLN-1080 ##STR00063## R/S-i-pent--Gal
GLN-1106 ##STR00064## 3-Th--Bz--Gal GLN-1107 ##STR00065##
2-Th--Bz--Gal GLN-1108 ##STR00066## 5-Cl-2-Th--Bz--Gal GLN-1109
##STR00067## 5-Im--Bz--Gal GLN-1110 ##STR00068## 5-OA--Bz--Gal
GLN-1111 ##STR00069## 5-Th--Bz--Gal GLN-0926 ##STR00070## Nic--Gal
GLN-1066 ##STR00071##
[0180] Rather than inhibiting cholinesterases, the pro-galantamines
referred to in the present document are substrates of the enzyme,
as is exemplarily demonstrated in FIG. 3.
[0181] The data of FIGS. 2 and 3 demonstrate that pro-galantamines
of the present invention do not act as efficient inhibitors of
cholinesterase, as was described in the earlier documents discussed
above. Instead, they are substrates of these enzymes. Similarly,
they also do not interact to the same extend as galantamine with
neuronal nicotinic acetylcholine receptors (FIG. 4).
[0182] The pro-galantamines of the present invention therefore
either do not interact, or only to a very limited extend, with the
established target molecules of galantamine, in particular
cholinesterases and neuronal nicotinic acetylcholine receptors. As
pro-drugs they therefore have rather limited, if any, efficacy as
cognition enhancers, and also produce only limited peripheral and
central side effects, as compared to galantamine (see further
below).
[0183] As is exemplified and demonstrated by pharmacokinetics in
mice (FIGS. 5a and 5b, Table 5), R1-benzoyl-galantamine displays an
unexpectedly high brain-to-blood concentration ratio
(R.sub.BB-proGal>19), a large initial concentration in the
brain, and it is only slowly cleaved to galantamine, as seen in the
delayed appearance of a galantamine peak in brain and blood. The
R.sub.BB-value is significantly larger than what was expected from
the log P value which probably is due to the slow cleavage of the
pro-drug in the brain and a depot effect thereby produced.
[0184] In Table 5, the key pharmacokinetic data of
benzoyl-galantamine, of several other R1-pro-galantamines and of
galantamine (for comparison) are listed.
TABLE-US-00005 TABLE 5 Pharmacokinetic data of several
R1-pro-galantamines in the mouse Gln number (for reference see
table 4) logP Co (Brain) R-Pro R-Gal 1062 3.0 4812 19.3 2.2 1067
2.8 4665 7.5 1.7 0979 2.5 3166 6.2 2.4 0993 3.7 2150 6.4 0.6 0978
2.2 1985 6.9 2.3 1076 2.4 1245 1.1 1.0 Gal 1.7 1741 1.2
[0185] Co is the highest pro-galantamine concentration (ng/ml)
achieved in mouse brain after injection of 3 mg/kg of
pro-galantamine. R-Pro is the brain-to-blood concentration ratio of
pro-galantamine, R-Gal that of galantamine under these experimental
conditions. For comparison, Co and R-Gal are also provided for i.v.
injection of the same amount of galantamine.
[0186] These data establish that only a particular selection of R1
substitutions is capable of producing the following advantageous
properties of pro-galantamines; high initial concentration in the
brain, large R.sub.BB, and slow enzymatic conversion to
galantamine. In addition (not shown in the table), the preferred
R1-prodrugs display little, if any side effects, as they are only
very slowly converted to galantamine, thereby largely protecting
them from acting as galantamine while being transported from the
site of administration to the sites of action in the brain.
[0187] These properties may have significant impact for the use of
these compounds as drugs in Alzheimer's disease and other brain
diseases. As is representatively shown in FIG. 5,
R1-pro-galantamines of the present invention display in ferrets
much less gastro-intestinal side effects than galantamine. Ferrets
were used in these studies as they are known to be particularly
sensitive to gastro-intestinal side effects. In addition to the
classical emetic responses to galantamine and other ChE inhibitors,
we recorded salivation (SA), shivering (SH), respiratory problems
(RP) and diarrhea (DI) at the levels "none; 0", "moderate; 0.5"
(behaviour observed at low frequency and/or at low intensity) and
"intense; 1.0" (behaviour observed frequently and/or continuously
and/or at high intensity), and summated the scores for the four
animals each used per drug dose in these studies.
[0188] The data depicted in FIG. 6 for the two pro-galantamines
demonstrate that their side effects profile in ferrets is much less
severe (5-6 times less) than that of the same dose of galantamine.
The advantageous side effects profile is probably due to the
reduced affinity of interaction of these R1-pro-galantamines with
cholinesterases and neuronal nicotinic acetylcholine receptors
(FIGS. 2, 4).
[0189] The advantages of enhanced transport of selected R1-produgs
through the blood-brain barrier into the brain, enzymatic
conversion to galantamine close to target sites in the central
nervous system, and interaction with such sites is producing
enhanced reversal of drug-induced amnesia in mice, as is shown in
FIG. 7 for three pro-glantamines (and for galantamine in
comparison).
[0190] The data of FIG. 7 suggest that Gln-1062 is approximately
4-times more potent than galantamine in reversing
scopolamine-induced amnesia in mice. It may be expected that a
similar or larger increase in drug efficacy can be achieved in man
when the particular R1-pro-galantamine is administered instead of
galantamine. The advantageous drug properties of
R1-pro-galantamines (higher efficacy, lesser or less intense side
effects) were also shown in other animal models.
[0191] In summary, the compounds of this invention are particularly
useful as medicaments for the treatment of human brain diseases
associated with a cholinergic deficit, including the
neurodegenerative diseases Alzheimer's and Parkinson's disease and
the neurological/psychiatric diseases vascular dementia,
schizophrenia and epilepsy. Based on preclinical studies using
various animal models, the compounds have dramatically reduced side
effects as compared to galantamine, including much fewer, if any,
incidents of emetic responses, diarrhea and vomiting. Moreover,
when enzymatically cleaved, the resulting galantamine displays an
advantageous pharmacokinetic profile in the brain and, due to its
enhanced concentration level in the brain, displays also enhanced
efficacy in interaction with the target molecules located in the
brain. Taken together, these properties make the administration of
galantamine as a R1-prodrug a preferred medication in the diseases
mentioned above.
Pharmaceutical Compositions and Administration
[0192] Acids useful for preparing the pharmaceutically acceptable
acid addition salts according to the invention include inorganic
acids and organic acids, such as sulfamic, amidosulfonic,
1,2-ethanedisulfonic, 2-ethylsuccinic, 2-hydroxyethanesulfonic,
3-hydroxynaphthoic, acetic, benzoic, benzenesulfonic acid,
carboxylic, ethylenediamine tetraacetic acid, camphorsulfonic,
citric, dodecylsulfonic, ethanesulfonic, ethenesulfonic,
ethylenediamine tetraacetic, fumaric, glubionic, glucoheptonic,
gluconic, glutamic, hexylresorcinic, hydrobromic, hydrochloric,
isethionoc, (bi)carbonic, tartaric, hydriodic, lactic, lactobionic,
laevulinic, laurylsulfuric, lipoic, malic, maleic, malonic,
mandelic, methanesulfonic, mucic, naphthalenesulfonic, nitric,
oxalic, pamoic, pantothenic, perchloric, phosphoric,
polygalacturonic, pectic, propionic, salicylic, succinic or
sulfuric acid, p-tuluenesulfonic, wherein hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric and perchloric acids, as
well as tartaric, citric, acetic, succinic, maleic, fumaric and
oxalic acids are preferred.
[0193] The active compounds of the present invention may be orally
administered, for example, with an inert diluent or with an edible
carrier, or they may be enclosed in gelatin capsules, or they may
be compressed into tablets. For the purpose of oral therapeutic
administration, the active compounds of the invention may be
incorporated with excipients and used in the form of tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, chewing
gum and the like. These preparations should contain at least 0.5%
of active compounds, but may be varied depending upon the
particular form and may conveniently be between 5% to about 70% of
the weight of the unit. The amount of active compound in such
compositions is such that a suitable dosage will be obtained.
Preferred compositions and preparations according to the present
invention are prepared so that an oral dosage unit form contains
between 0.1-50 milligrams of active compound.
[0194] The tablets, pills, capsules, troches and the like may also
contain the following ingredients: a binder such as
micro-crystalline cellulose, gum tragacanth or gelatin: an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, cornstarch and the like; a lubricant such
as magnesium stearate or Sterotex; a glidant such as colloidal
silicon dioxide; and a sweetening agent such as sucrose or
saccharin may be added or a flavouring agent such as peppermint,
methyl salicylate, or orange flavouring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the
above-type, a liquid carrier such as an oil. Other dosage unit
forms may contain other various materials which modify the physical
form of the dosage unit, for example, as coatings. Thus, tablets or
pills may be coated with sugar, shellac, or other enteric coating
agents. A syrup may contain, in addition to the active compounds,
sucrose as a sweetening agent and certain preservatives, dyes,
colourings and flavours. Materials used in preparing these various
compositions should be pharmaceutically pure and non-toxic in the
amounts used.
[0195] For the purpose of nasal or parenteral therapeutic
administration, the active compounds of the invention may be
incorporated into a solution or suspension. These preparations
should contain at least 0.1% of active compound, but may be varied
between 0.5 and about 30% of the weight thereof. The amount of
active compound in such compositions is such that a suitable dosage
will be obtained. Preferred compositions and preparations according
to the present inventions are prepared so that a nasal or
parenteral dosage unit contains between 0.1 to 20 milligrams of
active compound.
[0196] Further the compounds of the present invention can be
administered via intranasal delivery to the cerebral spinal fluid
as disclosed in detail in WO2004/02404.
[0197] The solutions or suspensions may also include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents, such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as ethylene-diamine
tetraacetic acid; buffers such as acetates; citrates or phosphates
and agents for the adjustment of tonicity such as sodium chloride
or dextrose. Parenteral multiple dose vials may be of glass or
plastic.
[0198] Typical dosage rates in administration of the active
ingredients depend on the nature of the compound that is used and
in intravenous administration are in the range of 0.01 to 2.0 mg
per day and per kilogram of body weight based on the physical
condition and other medications of the patient.
[0199] The following specific formulations exemplify suitable
applications: Tablets and capsules that contain 0.5 to 50 mg.
Solution for parenteral administration that contains 0.1 to 30 mg
of active ingredient/ml. Liquid formulations for oral
administration at a concentration of 0.1 to 15 mg/ml. Liquid
formulations for nasal or intra-cerebroventricular administration
at a concentration of 0.1 to 5 mg of active ingredient/ml. The
compounds according to the invention can also be administered by a
transdermal system, in which 0.1 to 10 mg/day is released. A
transdermal dosage system may consists of a storage layer that
contains 0.1 to 30 mg of the active substance as a free base or
salt, in case together with a penetration accelerator, e.g.,
dimethyl sulfoxide, or a carboxylic acid, e.g., octanoic acid, and
a realistic-looking polyacrylate, e.g., hexylacrylate/vinyl
acetate/acrylic acid copolymer including softeners, e.g.,
isopropylmyristate. As a covering, an active ingredient-impermeable
outside layer, e.g., a metal-coated, siliconised polyethylene patch
with a thickness of, for example, 0.35 mm, can be used. To produce
an adhesive layer, e.g., a dimethylamino-methacrylate/methacrylate
copolymer in an organic solvent can be used.
[0200] The invention also relates to pharmaceutical compositions
that in a pharmaceutically acceptable adjuvant contain a
therapeutically effective amount of at least one of the compounds
that are proposed according to the invention.
[0201] Examples of chemical synthesis and properties of derivatives
are given in the following examples. Abbreviations: DCM:
dichloromethane; DMAP: 4-dimethylaminopyridine; DCC:
dicyclohexylcarbodiimide; DCHU: dicyclohexylurea.
Example 1
[0202] N-Methoxymethyl-galanthaminiumchloride
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-11-methoxymethyl-11-methyl-6H-6--
hydroxy-3-methoxy-benzofuro[3a,3,2-ef][2]benzaze-pinium,
chloride).
[0203] N-Methoxymethyl-galanthaminiumchloride is obtained from
Galantamine via alkylation using chloromethylmethylether:
##STR00072##
[0204] To a solution of (-)-Galantamine (5.00 g, 17.4 mmol) in dry
dimethylformamide (12 mL) chloromethylmethylether (1.12 g, 13.9
mmol) is added at -5 bis 0.degree. C. in the course of 15 min and
stirred for 4 hrs. at room temperature. The reaction mixture is
poured on ethyl acetatet (500 mL) and the precipitate obtained is
filtered and washed using ethyl acetate (3.times.50 mL).
[0205] The crude product (4.20 g, 82%) has a purity of 96% (HPLC).
For further purification the crude product is dissolved in dry
ethanol, stirred after the addition of activated charcoal, filtered
and added to ethyl acetate (500 mL). The precipitate is filtered
and washed using ethyl acetate (3.times.50 mL) and dry diethylether
(1.times.50 mL). The product is obtained in the form of colourless
crystals (3.85 g, 75% d. Th.) melting at 126-127.degree. C.
[0206] Opt. Rotation: [.alpha.].sub.D.sup.20=-113.9.degree. (c=0.18
g/water) calcd. For C.sub.19H.sub.26ClNO.sub.4*0.33H.sub.2OC,
61.04; H, 7.19; N, 3.75. found: C, 61.10; H, 7.07; N, 3.75
[0207] .sup.1H NMR (DMSO-d6) .delta. 6.86 (s, 2H), 6.29 (d, J=10
Hz, 1H), 5.88 (d, J=10 Hz, J=4 Hz, 1H), 5.13 (bs, 3H), 4.66 (s,
2H), 4.48 (d, J=14 Hz, 1H), 4.22-3.90 (m, 2H), 3.81 (s, 3H), 3.70
(s, 3H), 3.70-3.52 (m, 1H), 2.75 (s, 3H), 2.44-1.79 (m, 4H);
.sup.13C NMR (DMSO-d6) .delta. 146.4 (s), 145.2 (s), 132.8 (s),
130.2 (d), 125.3 (d), 123.7 (d), 117.8 (s), 112.1 (d), 94.8 (t),
86.4 (d), 61.7 (d), 60.3 (t), 59.4 (q), 56.2 (t), 55.6 (q), 46.2
(s), 40.2 (q), 31.1 (2 t);
[0208] The chemical and biological stability of this compound has
been determined in various buffers (chemical stability), in rat
blood serum, and in rat brain extract, suggesting that the
derivative can act as a pro-drug.
[0209] Instead of chloromethyl or methyl ether the following
reagents can be used alternatively:
Methoxymethanolbenzenesulfonate, trifluoromethanesulfonic acid
methoxymethyl ester, or methoxymethanol
4-methylbenzenesulfonate.
Example 2
Tert-Butoxycarbonylamino-acetic acid (N-norgalanthaminyl)-methyl
ester
##STR00073##
[0211] To a solution of N-Boc-glycine chloromethylester (1.0 mmol)
and norgalantamine (1.0 mmol) in dry DMF (2.0 mL) triethylamine (3
mmol) was added dropwise and the reaction stirred under nitrogen
for 3 days. The triethylammonium chloride formed was filtered and
washed with dry ether and the filtrate rotoevaporated to dryness.
The residue was redissolved in dry acetone (2 ml) upon heating and
left to stand overnight at 4.degree. C. for additional
precipitation of the triethylammonium salt. After renewed
filtration and rotoevaporation the mixture was chromatographed on
silica using ethyl acetate/petrol ether. The target product was
isolated as an oil.
[0212] .sup.13H NMR (DMSO-d6) .delta. 28.5, 33.9, 37.9, 42.0, 48.2,
51.2, 56.2, 56.9, 61.9, 79.5, 79.9, 88.8, 111.8, 121.3, 126.6,
129.8, 130.8, 133.6, 145.8, 148.2, 156.3, 169.6.
Example 3
2-tert-Butoxycarbonylamino-3-phenylpropionic acetic acid
(N-norgalantha-minyl)-methyl ester
##STR00074##
[0214] This compound was prepared using the procedure of example 2
with N-Boc-phenylalanine chloromethylester.
[0215] .sup.13H NMR (DMSO-d6) .delta. 28.5, 33.9, 36.9, 37.9, 48.2,
51.2, 54.6, 56.2, 56.9, 61.9, 79.5, 80.2, 88.8, 111.8, 121.3,
126.0, 126.6, 127.8, 128.7, 129.8, 130.8, 133.6, 139.5, 145.8,
148.2, 156.0, 171.6.
Example 4
(3R,4aS,9bS)-9-Dimethylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-9b-vinyl-
-dibenzofuran-3-ol;
(=10,11-Seco-11,12-dehydro-10-methyl-galantamine)
##STR00075##
[0217] A solution of N-methylgalanthaminium iodide (5.0 g, 11.6
mmol) in 35% aqueous potassium hydroxide (150 mL) is heated under
reflux for 48 hrs, diluted with water (200 mL) and acidified using
conc. hydrochloric acid to pH=3-4 and extracted with
dichloromethane (2.times.50 mL) to remove non-basic compounds. The
aqueous phase is basified using conc. ammonia to pH 12 and
extracted using dichloromethane (4.times.100 mL). The combined
organic extracts are washed with brine (2.times.50 mL), dried using
sodium sulfate and rotoevaporated to obtain the crude product which
is purified by MPLC (200 g SiO.sub.2, chloroform:methanol=99:1+1%
conc. ammonia). The product is obtained as yellow oil (2.5 g, 71%
d. Th.). The fumarate (colourless crystals) and oxalate salt
(off-white crystals) where obtained in the usual way: m.p.:
151-153.degree. C. (fumarate), 116-118.degree. C. (oxalate).
[.alpha.].sub.D.sup.20=-56.5.degree. (0.212 g/100 mL H.sub.2O)
(fumarate).
fumarate:
[0218] C.sub.18H25.sub.NO.sub.3* 1.0 C.sub.4H.sub.4O.sub.4
[0219] Calcd.: C, 62.99; H, 6.97; N, 3.34
[0220] Found: C, 62.89; H, 6.62; N, 3.32
oxalate
[0221] C.sub.18H.sub.25NO.sub.3* 1.0 C.sub.2H.sub.2O.sub.40.75
H.sub.2O
[0222] Calcd.: C, 59.32; H, 6.60; N, 3.46
[0223] Found.: C, 59.48; H, 6.31; N, 3.38
[0224] .sup.1H-NMR (CDCl.sub.3): .delta. 6.83 (d, J=8.4 Hz, 1H),
6.72 (d, J=8.4 Hz, 1H), 6.13-5.95 (m, 3H), 5.32 (dd, J=10.3, 1.1
Hz, 1H), 5.25 (dd, J=18.3, 1.1 Hz), 4.63 (b, 1H), 4.15 (b, 1H),
3.85 (s, 3H), 3.58 (d, J=12.8 Hz, 1H), 3.07 (d, J=12.8 Hz, 1H),
2.56 (m, 1H), 2.15 (s, 6H), 1.96 (ddd, J=16.2, 4.9, 2.3 Hz, 1H);
.sup.13C-NMR (CDCl.sub.3): .delta. 146.6 (s), 144.1 (s), 139.0 (t),
132.2 (s), 128.6 (s), 128.1 (d), 127.8 (d), 123.6 (d), 117.3 (t),
111.1 (d), 86.0 (d), 62.0 (t), 59.7 (t), 55.7 (q), 52.9 (s), 44.7
(q), 28.6 (t)
Example 5
(3R,4aS,9bS)-6-Methoxy-9-methylaminomethyl-3,4,4a,9b-tetrahydro-9b-vinyl-d-
ibenzofuran-3-ol; (=10,11-Seco-11,12-dehydro-galantamine)
##STR00076##
[0226] 3-Chloroperbenzoic acid (0.38 g, 75% ig, 1.66 mmol) is added
to a solution of
(3R,4aS,9bS)-9-dimethylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-9b-viny-
l-dibenzofuran-3-ol (0.50 g, 1.66 mmol) in dichloromethane (35 mL)
and then stirred for 30 minutes at room temperature. After adding a
solution of iron(II)sulfate-heptahydrate (0.23 g, 0.83 mmol) in
methanol (5 mL) it is then stirred for another 20 minutes at room
temperature. Then 2N hydrochloric acid (30 mL) is added, stirred
for 5 minutes and most of the dichloromethane is removed by
rotoevaporation. The remaining aqueous phase is washed with diethyl
ether (4.times.20 mL), basified to pH 12 using concentrated ammonia
and then extracted with dichloromethane (4.times.40 mL). The
combined organic phases are washed with saturated sodium chloride
solution (30 mL), dried using sodium sulphate, filtered and the
solvent is again removed by rotoevaporation to obtain the crude
product which is then further purified using MPLC (Buchi, 110 g
SiO.sub.2, chloroform:methanol 97:3+1% concentrated ammonia) and
obtained as a yellow oil (0.30 g, 63% d. Th.). The oxalate is
prepared in the usual way and obtained as colourless crystals, 0.37
g, 59% d. Th., m.p. 127-129.degree.. The purity is checked by TLC
(chloroform:methanol=9:1+1% conc. ammonia, R.sub.f=0.35).
[.alpha.].sub.D.sup.20-41.8.degree. (0.220 g/100 mL H.sub.2O)
(Oxalate)
[0227]
C.sub.17H.sub.21NO.sub.3*1.0C.sub.2H.sub.2O.sub.4*0.5H.sub.2O
[0228] Calcd.: C, 59.06; H, 6.26; N, 3.62
[0229] Found: C, 59.35; H, 6.00; N, 3.56
[0230] .sup.1H-NMR (CDCl.sub.3): .delta. 6.88 (d, J=8.4 Hz, 1H),
6.75 (d, J=8.4 Hz, 1H), 6.15-5.82 (m, 3H), 4.67 (b, 1H), 4.09-4.20
(m, 1H), 3.85 (s, 3H), 3.68 (s, 2H), 2.52-2.49 (m, 1H), 2.42 (s,
3H), 1.97 (ddd, J=16.2, 4.9, 2.3 Hz, 1H); 13C-NMR (CDCl.sub.3):
.delta. 146.6 (s), 144.0 (s), 139.4 (d), 131.6 (s), 129.5 (s),
128.9 (d), 127.2 (d), 122.7 (d), 117.6 (t), 111.8 (d), 86.0 (d),
62.0 (d), 55.9 (q), 52.8 (t), 51.1 (s), 36.0 (q), 28.8 (t)
Example 6
(3R,4aS,9bS)-9-Dimethylaminomethyl-9b-ethyl-6-methoxy-3,4,4a,9b-tetrahydro-
-dibenzofuran-3-ol; (=10,11-Seco-10-methyl-galantamine)
##STR00077##
[0232] Palladium (10%) on active carbon (90 mg) is pre-hydrogenated
in methanol (40 mL) and conc. acetic acid (2 mL) in the
Parr-apparatus at 10 psi and room temperature for 45 minutes. After
adding
(3R,4aS,9bS)-9-dimethylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-9b-viny-
l-dibenzofuran-3-ol (0.90 g, 2.99 mmol) it is then hydrated for 8
hrs. at 15-20 psi and room temperature. The catalyst is then
filtered and the solvent is removed by rotoevaporation. The residue
is then dissolved in water (100 mL), basified using conc. ammonia
and extracted using dichloromethane (5.times.40 mL). The combined
aqueous phases are washed with a saturated sodium chloride solution
(2.times.20 mL), dried using sodium sulphate and the solvent is
removed by rotoevaporation. It is then further purified using MPLC
(Buchi, 110 g SiO.sub.2, chloroform:methanol=98:2+1% conc.
ammonia), obtained as a colourless oil (0.80 g, 88%) and converted
to the hydrochloride m.p. 248-249.degree..
[.alpha.].sub.D.sup.20=47.3.degree. (0.220 g/100 mL H.sub.2O). TLC
chloroform:methanol=9:1+1% conc. ammonia, R.sub.f=0.45.
[0233] C.sub.18H.sub.25NO.sub.3*2.0 HCl
[0234] Calcd.: C, 57.45; H, 7.23; N, 3.72
[0235] Found: C, 57.95; H, 6.85; N, 3.48
[0236] .sup.1H-NMR (CDCl.sub.3): .delta. 6.78 (d, J=8.3 Hz, 1H),
6.67 (d, J=8.3 Hz, 1H), 6.12 (d, J=10.2 Hz, 1H), 5.89 (dd, J=10.2,
4.3 Hz, 1H), 4.74 (b, 1H), 4.19-4.09 (m, 1H), 3.84 (s, 3H), 3.54
(d, J=12.9 Hz, 1H), 3.19 (d, J=12.9 Hz, 1H), 2.53-2.32 (m, 1H),
1.94-2.13 (m, 2H), 1.69 (ddd, J=16.2, 4.9, 2.3 Hz, 1H), 0.85 (t,
J=7.6 Hz, 3H); .sup.13C-NMR (CDCl.sub.3): .delta. 146.8 (s), 144.4
(s), 131.6 (d), 128.2 (s), 127.7 (d), 123.6 (d), 110.6 (d), 83.8
(d), 62.7 (d), 61.6 (s), 55.7 (q), 51.1 (s), 45.2 (q), 31.6 (t),
27.5 (t),
Example 7
3.3.4.
(3R,4aS,9bS)-9b-Ethyl-9-methylaminomethyl-6-methoxy-3,4,4a,9b-tetra-
hydro-dibenzofuran-3-ol; (=10,11-Seco-galantamine)
##STR00078##
[0238] Following the procedure of example 6 using
(3R,4aS,9bS)-9-Dimethylaminomethyl-9b-ethyl-6-methoxy-3,4,4a,9b-tetrahydr-
o-dibenzofuran-3-ol the pure product is obtained as a yellow oil
(0.17 g, 59% d. Th.) and converted to the oxalate and fumarate.
[0239] M.p. (oxalate) 162-164.degree.,
[.alpha.].sub.D.sup.20=-51.2.degree. (0.146 g/100 mL H.sub.2O)
(oxalate).
[0240] TLC chloroform:Methanol=9:1+1% conc. ammonia,
R.sub.f=0.39
fumarate:
[0241] C.sub.17H.sub.23NO.sub.3* 1 C.sub.4H.sub.4O.sub.4* 0.33
H.sub.2O
[0242] Calcd.: C, 61.31; H, 6.78; N, 3.40
[0243] Found: C, 61.22; H, 6.67; N, 3.32
oxalate
[0244] C.sub.17H.sub.23NO.sub.3* 1 C.sub.2H.sub.2O.sub.40.25
H.sub.2O
[0245] Calcd.: C, 59.44; H, 6.69; N, 3.65
[0246] Found.: C, 59.43; H, 6.78; N, 3.65
[0247] .sup.1H-NMR (CDCl.sub.3): .delta. 6.85 (d, J=8.4 Hz, 1H),
6.73 (d, J=8.4 Hz, 1H), 5.97-5.92 (m, 2H), 4.74 (dd, J=5.8, 3.5 Hz,
1H), 4.22-4.12 (m, 1H), 3.84 (s, 3H), 3.74 (d, J=7.2 Hz, 2H), 2.48
(s, 3H), 2.45-2.28 (m, 2H), 2.20-1.62 (m, 5H), 0.85 (t, J=7.46,
3H); .sup.13C-NMR (CDCl3): .delta. 146.6 (s), 144.2 (s), 131.1 (s),
131.0 (d), 128.9 (s), 128.8 (d), 122.3 (d), 111.1 (d), 83.8 (d),
62.8 (d), 55.8 (q), 51.0 (s), 36.3 (q), 32.5 (t), 29.1 (t),
Example 8
(4aS,6R,.sup.8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofu-
ro[3a,3,2-ef][2]benzazepin-6-yl-.beta.-D-glucopyranosiduronic acid
(=Galantamine-3-glucuronide)
##STR00079##
[0248] Step 1: Methyl
1,2,3,4-tetra-O-isobutyryl-.beta.-D-glucopyranuronate (2)
[0249] To a solution of NaOMe (26 mg, 0.48 mmol) in MeOH (150 mL)
was added glucurono-6,3-lactone (20.6 g, 154 mmol) in portions with
stifling until dissolved. The solvent was then removed in vacuo,
the residue taken up in pyridine (85 mL, 1.08 mol) and the solution
cooled to 0.degree. C. Isobutyryl chloride (110 mL, 1.06 mol) in
CH.sub.2Cl.sub.2 (70 mL) was then added with strong mechanical
stirring at a rate that kept the temperature below 10.degree. C.,
and the reaction mixture was left at room temperature overnight.
More CH.sub.2Cl.sub.2 (100 mL) was then added and the solution
washed with water (400 mL), 2 M HCl (3.times.50 mL), saturated
sodium bicarbonate (5.times.50 mL) and brine (50 mL). After drying,
filtering and evaporating in vacuo, a gum was obtained which
crystallized on trituration with petroleum ether (40-60.degree.
C.). Filtration and drying at 40.degree. C. in a vacuum oven
yielded the title product. Recrystallisation from MeOH or petrol
ether afforded the pure .beta. isomer 2 as needles, mp 127.degree.
C., (21.6 g, 37%, from mother liquid some more product could be
isolated) [.alpha.].sub.D=+11.12 (c 1.7 CHCl3); .delta..sub.H (300
MHz, CDCl.sub.3): 5.78 (d, J=8 Hz), 5.39 (t, J=9.5 Hz), 5.25 (t,
J=9.5 Hz), 5.23 (dd, J=9.5, 8 Hz), 4.19 (d, J=9.5 Hz), 3.75 (s,
OMe), 2.65-2.45 (m, 4.times.CHMe.sub.2), 1.17-1.07 (m,
4.times.CHMe.sub.2).
[0250] An alternative procedure with pivaloyl chloride was also
used to prepare methyl
1,2,3,4-tetra-O-pivaloyl-.beta.-D-glucopyranuronate in 21% (the
isolation and crystallization of compound 2 was easier).
Step 2: Methyl 2,3,4-tri-O-isobutyryl-D-glucopyranuronate (3)
[0251] Ammonia gas pre-dried by passing it through a bed of sodium
hydroxide was bubbled through CH.sub.2Cl.sub.2 (200 mL) at
-4.degree. C. over 1 h at a rate which kept the temperature below
0.degree. C. The above methyl
1,2,3,4-tetra-O-isobutyryl-.beta.-D-glucopyranuronate (3.0 g, 8
mmol) was added and the solution stirred at 0.degree. C. for 3 h
and then left at room temperature for 20 h. Nitrogen gas was
bubbled through the solution for 30 min. and it was extracted with
ice-cold 10% aqueous HCl, then water. The organic phase was dried
over Na.sub.2SO.sub.4 filtered and solvent removed in vacuo to
leave the crude product. Recrystallization from CHCl.sub.3:PE
afforded the pure microcrystalline .alpha.-epimer, mp 89.degree. C.
.delta.H (300 MHz, CDCl.sub.3): 5.65 (t, J=10 Hz), 5.54 (d, J=3.5
Hz), 4.92 (dd, J=10, 3.5 Hz), 4.60 (d, J=10 Hz), 3.75 (s, OMe),
2.61-2.43 (m, 4.times.CHMe.sub.2), 1.20-1.05 (m,
4.times.CHMe.sub.2).
Step 3: Methyl
2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-.alpha.-D-glucopyranurona-
te (4)
[0252] To a stirred solution of methyl
2,3,4-tri-O-isobutyryl-D-glucopyranuronate 3 (418 g, 1 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was added trichloroacetonitrile (0.4 mL,
3.7 mmol), followed by anhydrous potassium carbonate (83 mg, 0.6
mmol), and the mixture stirred for 40 h. It was filtered through a
short pad of silica and eluted with ether. Filtration and
evaporation in vacuo then yielded the title product 4 as a semi
crystalline gum which crystallized from dry isopropanol as white
prisms, mp 108.degree. C. (422 mg, 75%). .delta.H (300 MHz, CDCl3):
8.72 (s, NH), 6.66 (d, J=3.5 Hz), 5.70 (t, J=10 Hz), 5.30 70 (t,
J=10 Hz), 5.20 (dd, J=10, 3.5 Hz), 4.51 (d, J=10 Hz), 3.75 (s,
OMe), 2.60-2.43 (m, 3.times.CHMe.sub.2), 1.17-1.06 (m,
3.times.CHMe.sub.2).
Step 4: Galantamine-6-methyl
2,3,4-tri-O-isobutyryl-.beta.-D-glucopyranuronate (5)
[0253] A suspension of dried galantamine hydrobromide (92 mg, 0.25
mmol) and the above methyl
2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-.beta.-D-glucopyranuronat-
e 4 (282 mg, 0.5 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) containing 4
.ANG. molecular sieves was stirred under argon at room temperature,
while BF.sub.3.Et.sub.2O (0.1 mL, 0.5 mmol) was added. After 1 h,
virtually all of the starting materials had dissolved and stifling
was continued for 2 days. More CH.sub.2Cl.sub.2 (20 mL) was added,
the solution washed with saturated aq. sodium bicarbonate (10 mL),
water and brine before being dried. Filtration and evaporation in
vacuo afforded a semisolid residue, which was purified with MPLC on
silica. Elution with CHCl.sub.3/MeOH 97:3-20 gave 75 mg of the
glucuronide. Trituration with EtOH yielded 30 mg of pure 5.
Step 5:
(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benz-
ofuro[3a,3,2-ef][2]-benzazepin-6-yl-.beta.-D-glucopyranosiduronic
acid (Galantamine-3-glucuronide) (6)
[0254] 2M-NaOH (2.0 mL) was added to a stirred suspension of the
glucuronate 5 (30 mg) in MeOH (4 mL), and the mixture left
overnight. The solution was then acidified with glacial acetic acid
to pH 5.5, the solvent evaporated and purified over silica with
CHCl.sub.3: MeOH (saturated with dry NH.sub.3) 95:5. The
product-fraction was freeze-dried to afford 14 mg of 6 as a white
powder, m.p. 238.degree. (dec.).
[0255] .sup.1H NMR (MeOD, 200 MHz): 1.63-1.73 (m, 1H), 2.02-2.21
(m, 2H), 2.38 (s, 3H), 2.43-2.53 (m, 1H), 2.99-3.06 (m, 1H),
3.19-3.33 (m, 1H), 3.47-3.49 (m, 1H), 3.65-3.71 (d, 1H, J=14.9 Hz),
3.78 (s, 3H), 4.05-4.13 (d, 1H, J=14.9 Hz), 4.58 (m, 1H), 5.85-5.94
(dd, 1H, J2=4.8 Hz, J2=10.2 Hz), 6.15-6.21 (d, 1H, J2=10.2 Hz),
6.63-6.77 (m, 2H)
[0256] .sup.13C NMR (MeOD, 200 MHz): 23.22, 28.65, 34.57, 42.02,
43.33, 48.09, 54.03, 55.64, 60.43, 88.54, 112.18, 122.30, 127.16,
127.70, 128.64, 133.49, 144.65, 146.39
Example 9
Galantamine-3,6-di-.beta.-D-glucuronide
##STR00080## ##STR00081##
[0257] Step 1: Galantamine-3,6-di(methyl
2,3,4-tri-O-isobutyryl-.beta.-D-glucopyranuronate) (7)
[0258] Following the procedure for the preparation of
Galantamine-6-methyl
2,3,4-tri-O-isobutyryl-.beta.-D-glucopyranuronate but using
sanguinine (137 mg, 0.5 mmol) and the above imidate 4 (1.12 g, 2
mmol) in dry CH.sub.2Cl.sub.2 (10 mL) afforded, after analogous
workup a semisolid residue, that was purified with MPLC on silica.
Elution with CHCl.sub.3/MeOH 97:3-20 gave the crude product (180
mg). Trituration with EtOH yielded 130 mg of the pure product
7.
Step 2: Galantamine-3,6-.beta.-D-diglucuronide (8)
[0259] 2M-NaOH (2.0 mL) was added to a stirred suspension of the
above glucuronate 7 (130 mg) in MeOH (4 mL), and the mixture left
overnight. The solution was then acidified with glacial acetic acid
to pH 5.5, the solvents removed by freeze drying and the product
chromatographed on silica using CHCl.sub.3: MeOH (saturated with
dry NH.sub.3) 95:5. gave 48 mg (63.5%) of the product 8.
[0260] .sup.1H NMR (CDCl.sub.3, 200 MHz): 1.60-1.72 (m, 2H),
1.82-2.6 (m, 10H), 2.88-3.30 (m, 3H), 3.50-3.67 (m, 6H), 3.80-4.20
(m, 3H), 4.30-4.70 (m, 1H), 4.94-5.30 (m, 6H), 5.76-6.21 (m, 2H),
6.42-6.56 (m, 1H), 6.74-6.86 (m, 1H)
Example 10
3-Nicotinoyl-galantamine
##STR00082##
[0262] A solution of galantamine (431 mg, 1.5 mmol) in dry pyridine
(25 mL) was treated with nicotinoyl chloride (240 mg, 1.7 mmol) and
4-N,N-dimethylaminepyridine (5 mg) at 0.degree. and the solution
stirred to room temp. for 2 hrs. followed by heating to 45.degree.
for 1 hr. The reaction mixture was poured on water (150 mL) and the
pH adjusted to 8.0 followed by extraction with dichloromethane. The
organic extract was washed with water and brine, dried (sodium
sulphate) and evaporated to give the crude product (480 mg,
81.5%)
[0263] .sup.13H NMR (DMSO-d6) .delta. 27.7, 34.3, 41.7, 47.8, 53.6,
55.9, 60.3, 63.2, 86.2, 111.5, 121.3, 122.1, 122.7, 126.0, 129.2,
130.6, 131.9, 136.4, 143.9, 146.5, 150.4, 151.5, 166.0.
[0264] This product was converted to the dihydrobromide salt by
dissolution in a minimum amount of warm 40% hydrobromic acid
followed by cooling and obtained as colorless crystals.
[0265] Anal. calcd. for C.sub.23H.sub.24N.sub.2O.sub.4. 2 HBr.
0.33H.sub.2.degree. C. 49.31; H, 4.80; N, 5.00. Found C, 49.10; H,
5.05; N, 4.85.
Example 11
(+-)-8-fluorogalantamine
##STR00083##
[0266] Step 1: 2-Fluoro-5-hydroxy-4-methoxy benzaldehyde (1)
[0267] Sulphuric acid (50 ml, 95-98%) was heated with stirring to
the 90-95.degree. C. under a dry nitrogen and
4,5-dimethoxy-2-fluoro benzaldehyde (10.1 g, 54.8 mmol) added
quickly and this mixture was stirred at the same temperature for
3.5 h. Reaction was followed by HPLC and found to be complete after
this time. The reaction mixture was poured on crushed ice (150 g)
and the white slurry obtained was heated to 65.degree. C. and
allowed to cool in the fridge overnight. The white precipitate was
filtered and washed with water (2.times.100 ml). The wet cake was
dried in the desiccator under reduced pressure to afford the
product (7.6 g, 82%, HPLC 95%, m.p.: 146-148) as off white
crystals.
Step 2:
4-Fluoro-5-{[2-(4-hydroxyphenyl)ethylamino]-methyl]-2-methoxy-phen-
ol (2)
[0268] A solution of 1 (7.6 g, 45 mmol) and tyramine (6.7 g, 49
mmol) in dry toluene (250 ml) and n-butanol (250 ml) was heated and
stirred to reflux for 5 h on the Dean-Stark apparatus to remove the
water. Reaction development was controlled by TLC
(MeOH:CH.sub.2Cl.sub.2 1:9) and reaction was found to be complete
after this time. Solvents were rotoevaporated and residue was
dissolved in dry methanol (500 ml). NaBH.sub.4 (1.8 g, 45 mmol) was
added at the temperature 0-5.degree. C. and this mixture was
stirred overnight while the temperature was raised to room
temperature and a white solid precipitated from the reaction
mixture. The solid was filtered and washed with cold methanol
(2.times.50 ml). The white, wet cake was dried in the desiccator at
reduced pressure to give the product (9.6 g, 74%, HPLC >99%) as
a white powder. The filtrate was rotaevaporated to give a brown
slurry (3.6 g), which was chromatographed on silica
(dichloromathane/methanol, gradient 0-10%) to give another (2.5 g,
19%, HPLC >99%) of product as a off white powder (total yield
93%, m.p.: 160-162.degree. C.).
[0269] .sup.1H NMR (MeOD, 200 MHz): 2.69 (s, broad, 4H), 3.66 (s,
2H), 3.80 (s, 3H), 6.66-6.77 (m, 4H), 6.96-7.00 (m, 2H).
Step 3:
N-[(2-fluoro-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphen-
yl)ethyl]-formamide (3)
[0270] To a suspension of 2 (7.63 g, 26.1 mmol) in dioxane (50 ml)
a solution of ethyl formiate (3.1 ml, 37.7 mmol), DMF (1.5 ml) and
formic acid (0.25 ml, 6.62 mmol) was added dropwise and the
reaction mixture was heated under argon to reflux for 10 h. The
reaction development was controlled by HPLC and showed complete
conversion after this time. Volatiles were removed under reduced
pressure, the residue was dissolved in methanol (32 ml) and poured
on crushed ice (160 ml), the white precipitate formed was stirred
magnetically for 1 h, filtered, washed with water (3.times.100 ml)
and dried to weight to afford the product (6.8 g, 81.3%, HPLC
>99%, m.p.: 153-168.degree. C.) as a white powder.
[0271] .sup.1H NMR (DMSO, 200 MHz): 2.49-2.67 (m, 2H), 3.15-3.29
(m, 2H), 3.75 (s, 3H), 4.28-4.35 (d, 2H, J.sub.2=13.89 Hz),
6.64-6.95 (m, 6H), 7.84 (s, 0.5H), 8.20 (s, 0.5H), 8.95-9.00 (d,
1H, 10.17 Hz), 9.18-9.20 (d, 1H, J=2.44 Hz).
Step 4:
4.alpha.,5,9,10,11,12-Hexahydro-1-fluoro-3-methoxy-11-formyl-6H-be-
nzofuro[3a,3,2-ef]benzazepine-6-one (4)
[0272] To the vigorously stirred biphasic mixture of potassium
carbonate (13.2 g, 95.5 mmol) and potassium hexacyanoferrate (28 g,
85.4 mmol) in toluene (580 ml) and water (120 ml), preheated to
50.degree. C., finely pulverized 3 (6.83 g, 21.4 mmol) was added in
one portion and this suspension was heated at 50-60.degree. C. with
intense stirring for 1 h. After this time the reaction mixture was
filtered trough the pad of celite, the toluene phase separated and
the water phase was extracted with toluene (2.times.100 ml). The
combined organic phases were dried (Na.sub.2SO.sub.4) and
roto-evaporated under reduced pressure to afford the product (1.3
g, 19%, HPLC 98%) as a white powder.
[0273] .sup.1H NMR (DMSO, 200 MHz): 1.75-1.93 (m, 1H), 2.15-2.30
(m, 1H), 2.73-2.83 (m, 1H), 3.00-3.12 (m, 1H), 3.40 (s, 4H),
3.98-4.13 (m, 1H), 4.28-4.35 (m, 0.5H), 4.51-4.97 (m, 2H),
5.27-5.34 (d, 0.5H, J=15.45 Hz), 5.94-6.00 (d, 1H, J=10.37 Hz),
6.77-6.86 (m, 1H), 7.15-7.26 (m, 1H), 8.10-8.15 (d, 1H, J=8.99
Hz)
[0274] .sup.13C NMR (DMSO, 200 MHz): 34.02, 37.21, 37.32, 45.45,
49.33, 49.53, 56.05, 87.29, 100.20, 100.34, 100.77, 100.90, 114.55,
114.93, 115.08, 126.66, 130.83, 130.93, 143.12, 143.43, 143.64,
143.76, 144.29, 144.52, 162.39, 162.62, 194.77.
Step 5:
1-Bromo-4a,5,9,10-tetrahydro-3-methoxy-spiro[6H-benzofuro[3a,3,2-e-
f][2]benzazepine-6,2'-[1,3]dioxolane]-11(12H)-carboxaldehyde
(5)
[0275] To the solution of 4 (1.084 g, 3.42 mmol) in toluene (10 ml)
a solution of 4-toluene sulphonic acid (0.02 g, 0.116 mmol) in
1,2-propane-diol (1.13 ml) was added and the mixture heated to the
reflux for 1 h while the water was removed using a Dean-Stark
apparatus. Another portion of 4-toluene sulphonic acid (0.05 g) in
1,2-propanediol (0.65 ml) was added and heating continued for
another 5 h. Reaction development was controlled by HPLC and the
reaction found to be complete after this time. The reaction mixture
was cooled to room temperature and extracted with acetic acid
(2.times.25 ml, 10% in water), sodium hydrogen carbonate
(2.times.25 ml, 10% in water) and brine (1.times.25 ml). The
toluene solution was dried (Na.sub.2SO.sub.4) and evaporated to
give a crude product (1.32 g) as an amber oil. This was
crystallized using i-propanol and ligroin to give product (0.92 g,
72%), as a colourless crystals.
[0276] .sup.1H NMR (CDCl.sub.3, 200 MHz): 0.74-2.66 (m, 10H),
2.98-4.86 (m, 8H), 5.44-5.74 (m, 1H), 6.34-6.39 (m, 1H), 7.98-8.03
(m, 1H).
(+-)-8-Fluoro-Narwedin (6)
[0277] To the solution of 5 (0.91 g, 2.43 mmol) in dry THF (15 ml)
lithium aluminium hydride (1.21 ml, 2.3 mol suspension in THF) was
added at 0-5.degree. C. under a continuous stream of dry nitrogen
and this mixture was stirred for 1 h. Another portion of lithium
aluminium hydride (0.605 ml, 2.3 mmol suspension in THF) was added
and stirring continued for additional 1 h while the temperature
raised slowly to room temperature. Reaction development was
controlled by HPLC and no starting material was detected after this
time. The reaction mixture was quenched with water/THF 1:1 (20 ml)
and volatiles removed under reduced pressure. The residue was
dissolved in 2N-hydrochloric acid (25 ml) and stirred at room
temperature for 30 min. The clear solution was than treated with
ammonia to pH 12 and extracted with ethyl acetate (3.times.50 ml).
The combined organic phases were dried (Na.sub.2SO.sub.4), treated
with charcoal, filtered and evaporated to dryness to give 720 mg of
the crude product as an brown oil. Chromatography on silica using 7
N NH.sub.3 in MeOH:CH.sub.2Cl.sub.2 5:95 as solvents afforded the
product (590 mg, yield 80%, HPLC 97%) as an amber oil.
[0278] .sup.1H NMR (CDCl.sub.3, 200 MHz): 1.77-1.84 (m, 1H),
2.09-2.24 (m, 1H), 2.38 (s, 3H), 2.60-2.71 (m, 1H), 2.98-3.11 (m,
3H), 3.64-3.72 (m, 4H), 4.03-4.11 (d, 1H, J=15.65 Hz), 4.65 (s,
1H), 5.93-5.98 (d, 1H, J=10.56 Hz), 6.40-6.46 (d, 1H, J=11.34 Hz),
6.84-6.89 (m, 1H)
[0279] .sup.13C NMR (CDCl.sub.3, 200 MHz): 33.41, 37.22, 43.18,
49.42, 49.46, 51.91, 51.99, 54.13, 56.20, 88.12, 99.99, 100.58,
114.98, 115.34, 127.31, 131.33, 131.43, 142.84, 143.51, 143.71,
144.11, 152.42, 157.18, 194.13.
(+-)-8-fluorogalantamine (7)
[0280] To the solution of 6 (500 mg, 1.64 mmol) in dry THF (30 ml)
L-Selectride (1.50 ml, 1 M solution in THF) was added dropwise at
-5 to 0.degree. C. under dry nitrogen and this mixture was stirred
at the same temperature for 30 min. The reaction was monitored by
HPLC and no starting material was detected after this time. The
reaction was quenched using water/THF 2:1 (50 ml) and solvents were
removed under reduced pressure. The residue was dissolved in
2N-hydrochloric acid (100 ml) and kept overnight in the fridge. The
aqueous solution was than washed with diethyl ether (2.times.30 ml)
and ammonia was added to pH 12. The aqueous phase was extracted
using ethyl acetate (3.times.100 ml), the combined organic phases
were washed with brine (50 ml), dried (Na.sub.2SO.sub.4) and
evaporated to afford the crude product (515 mg) as a clear,
slightly yellow oil which was purified by chromatography on silica
using MeOH:CH.sub.2Cl.sub.2 9:1 to afford the product (0.46 g, 92%,
HPLC >99%) as a white powder.
[0281] .sup.1H NMR (CDCl.sub.3, 400 MHz): 1.25 (s, 1H), 1.55-1.67
(m, 1H), 1.92-2.10 (m, 2H), 2.41 (s, 4H), 2.62-2.70 (m, 1H),
2.98-3.29 (m, 2H), 3.72-3.78 (d, 1H), 3.81 (s, 3H), 4.07-4.20 (m,
2H), 4.60 (s, 1H), 6.03 (s, 2H), 6.47-6.49 (d, 1H),
[0282] .sup.13C NMR (CDCl.sub.3, 400 MHz): 30.11 (C-5), 34.31
(C-9), 43.10 (N--CH.sub.3), 49.21 (C-8a), 52.15 (C-10), 54.32
(OCH.sub.3), 56.55 (C-12), 62.37 (C-6), 89.29 (C-4a), 99.86 (C-2),
100.16 (C-12a), 126.89 (C-12b), 134.25 (C-8), 134.30 (C-7), 142.09
(C-3a), 144.23 (C-3), 154.31 (C-1), 156.69 (C-1).
(-)-8-fluorogalantamine
[0283] The enantiomers of (+-)-8-fluorogalantamine were separated
using chiral preparative column chromatography (Chiracel OD, 5
.mu.m, 5 50 cm, 80% n-heptane/20% i-PrOH) to afford two isomers
which were converted to the corresponding hydrobromide salts. The
progress and the result of this chiral separation was analyzed by
chiral HPLC (Chiracel I OD-H, 80% n-heptane+0.1% diethyl amine/20%
i-PrOH). The crystal structure of (-) 2.HBr was determined thus
confirming the expectation, that (-)-8-fluorogalanthamine has the
same absolute configuration as (-)galantamine.
Example 12
Galantamine, 2-propylpentanoate (ester)
[0284] (-)Galantamine (287 mg, 1 mmol), 2-propyl-pentanoic acid
(216 mg, 1.5 mmol), 4-dimethylaminopyridine (244 mg, 2 mmoles) are
added to dry CH.sub.2Cl.sub.2 and stirred for 5 min. A solution of
dicyclohexylcarbodiimide (DCC, 2 ml of a 1M solution in
CH.sub.2Cl.sub.2) was added in increments and the mixture stirred
for 20 h under argon. After completion of reaction (as determined
by TLC, MeOH/CH.sub.2Cl.sub.2 10:90, visualization with molybdato
phosphoric acid) the precipitate was filtered using Hyflo
(=diatomaceous earth) and the filtrate was washed with 10%
NaHCO.sub.3 and water. The organic phase was evaporated and the
crude product obtained purified by preparative chromatography using
a gradient of 0 to 8% methanol and methylene chloride with UV
detection. The pure product was isolated by evaporation of the
appropriate fractions as a white solid.
[0285] .sup.1H NMR (CDCl.sub.3): 0.84 (6H, m); 1.28 (6H, m); 1.57
(3H, m); 2.20 (6H, m); 2.52 (1H, m); 3.11 (1H, m); 3.42 (1H, m);
3.79 (4H, m); 4.28 (1H, m); 4.56 (1H, m); 5.31 (1H, m); 5.91 (1H,
m); 6.32 (1H, m); 6.59 (2H, q).
[0286] Following the same procedure the following examples were
prepared:
TABLE-US-00006 ##STR00084## Example No. R .sup.1H NMR (CDCl.sub.3)
13 ##STR00085## 1.38 (9H, s); 1.67 (1H, m); 2.11 (2H, m); 2.48(3H,
s); 2.59 (1H, m); 2.97 (2H, m); 3.29(2H, m); 3.78 (3H, s); 3.65
(2H, m); 4.10 (1H, m); 4.44 (1H, m); 5.31 (1H, m); 5.81 (1H, m);
6.34 (1H, m); 6.61 (2H, m); 7.19 (5H, m) 14 ##STR00086## 1.31 (9H,
s); 1.54 (1H, m); 1.96(2H, m); 2.32(3H, s); 2.56 (1H, m); 3.01 (2H,
m); 3.30(2H, m); 3.78 (3H, s); 3.65 (2H, m); 4.08 (1H, m); 4.42
(1H, m); 5.23 (1H, m); 5.38 (2H, m); 5.79 (1H, m); 6.28 (1H, m);
6.51 (2H, m); 7.13 (8H, m) 15 ##STR00087## 1.41 (2H, m); 1.61 (4H,
m); 1.81 (1H, m); 2.03 (2H, m); 2.38 (6H, m); 2.69 (1H, m); 3.09
(3H, m); 3.30 (1H, m); 3.70 (1H, m); 3.83 (3H, s); 4.08 (1H, m);
4.55 (1H, m); 5.29 (1H, m); 5.90 (1H, q); 6.31 (1H, d), 6.63 (2H,
m) 16 ##STR00088## 1.49 (9H, m): 1.54 (9H, m); 2.13 (2H, m); 2.49
(4H, m); 2.63 (1H, m); 3.17 (4H, m); 3.78 (5H, m); 4.57 (2H, m);
5.33 (1H, m); 6.07 (1H, m); 6.31 (1H, m); 6.63 (2H, q); 7.27 (2H,
m) 17 ##STR00089## 1.03 (3H, t); 1.54 (1H, m); 2.00 (2H, m); 2.25
(2H, m); 2.55 (3H, s); 2.64 (1H, m); 2.94 (1H, m); 3.02 (1H, m);
3.54 (1H, m); 3.76 (3H, s); 4.09 (1H, m); 4.48 (1H, m); 5.26 (1H,
m); 5.76 (1H, m); 6.19 (1H, m); 6.56 (2H, q) 18 ##STR00090## 1.06
(6H, m); 1.41 (1H, m); 1.59 (1H, m); 2.05 (2H, m); 2.32 (3H, m);
2.53 (1H, m); 3.01 (1H, m); 3.23 (1H, m); 3.55 (1H, m); 3,78 (3H,
s); 4.09 (1H, m); 4.48 (1H, m); 5.22 (1H, m); 5.83 (1H, m); 6.47
(1H, d); 6.60 (2H, q) 19 ##STR00091## 1.07 (9H, m); 1.51 (1H, m);
2.00 (2H, m); 2.26 (2H, m); 2.59 (3H, s); 2.54 (1H, m); 2.98 (1H,
m); 3.02 (1H, m); 3.54 (1H, m); 3.76 (3H, s); 4.09 (1H, m); 4.48
(1H, m); 5.26 (1H, m); 5.81 (1H, m); 6.21 (1H, m); 6.62 (2H, q)
Example 20
L-Phenylalanine, N-[(1,1-dimethylethoxy)carbonyl]-,
(4aS,6S,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3-
a,3,2-ef][2]benzazepin-6-yl ester
[0287] To solution of (-) galantamine (287 mg, 1.0 mmol) in dry
CH.sub.2Cl.sub.2 (30 mL) N-Boc-phenylalanine (400 mg, 1.5 mmol) and
triphenyl phosphine (340 mg, 1.3 mmol) are added with magnetic
stirring followed by the drop-wise addition of diisopropyl
azodicarboxylate (DIAD) (270 mg, 1.34 mmoles.) to the reaction
mixture at -10.degree. C. The reaction was stirred overnight at
room temperature under argon. After the completion of the reaction
(TLC-MeOH/CH.sub.2Cl.sub.2 (10:90)) the reaction mixture was
filtered and the filtrate was washed with 10% NaHCO.sub.3 and
water. The organic phase was evaporated and the crude product
obtained purified by preparative chromatography using a gradient of
0 to 8% methanol and methylene chloride with UV detection. From the
fractions containing the pure products these were isolated by
evaporation of the solvents. This procedure results in the
inversion of configuration on oxygen in position 6.
[0288] .sup.1H NMR (CDCl.sub.3) 1.35 (9H, s); 1.60 (1H, m); 2.05
(2H, m); 2.36 (3H, s); 2.59 (1H, m); 2.90 (2H, m); 3.30 (2H, m);
3.58 (3H, s); 3.65 (2H, m); 4.08 (1H, m); 4.42 (1H, m); 5.23 (1H,
m); 5.79 (1H, m); 6.28 (1H, m); 6.54 (2H, m); 7.13 (5H, m)
Example 21
L-Phenylalanine,
(4aS,6S,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3-
a,3,2-ef][2]benzazepin-6-yl ester
[0289] L-Phenylalanine,
(4aS,6S,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3-
a,3,2-ef][2]benzazepin-6-yl ester was prepared from the compound
obtained in example 20 by Boc-deprotection using trifluoro acetic
acid in methylene chloride followed by the usual workup and
resulted in the product as a white powder.
[0290] .sup.1H NMR (CDCl.sub.3) 1.82 (2H, m); 2.05 (2H, m); 2.36
(3H, s); 2.59 (1H, m); 2.90 (2H, m); 3.30 (2H, m); 3.58 (3H, s);
3.65 (2H, m); 4.08 (1H, m); 4.42 (1H, m); 5.23 (1H, m); 5.79 (1H,
m); 6.28 (1H, m); 6.54 (2H, m); 7.13 (5H, m)
Example 22
L-tyrosine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-b-
enzofuro[3a,3,2-ef][2]benzazepin-6-yl ester
[0291]
L-tyrosine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-meth-
yl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-yl ester was prepared
from the compound of example 13 using the same deprotection method
as in example 21.
[0292] .sup.1H NMR (CDCl.sub.3) 1.68 (2H, m); 1.96 (2H, m); 2.32
(3H, s); 2.56 (1H, m); 3.01 (2H, m); 3.30 (2H, m); 3.78 (3H, s);
3.65 (2H, m); 4.08 (1H, m); 4.42 (1H, m); 5.23 (1H, m); 5.79 (2H,
m); 6.28 (1H, m); 6.51 (2H, m); 7.13 (4H, dd)
Example 23
L-histidine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H--
benzofuro[3a,3,2-ef][2]benzazepin-6-yl ester hydrochloride
[0293]
L-histidine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-met-
hyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-yl ester hydrochloride
was prepared from the compound of example 14 using HCl in ethyl
acetate for deprotection and resulted in the isolation of the
product as the hydrochloride.
[0294] .sup.1H NMR (CDCl.sub.3) 2.34 (2H, m); 2.54 (4H, m); 2.78
(1H, m); 3.21 (4H, m); 3.79 (5H, m); 4.58 (2H, m); 5.41 (1H, m);
6.18 (1H, m); 6.48 (1H, m); 6.65 (2H, q); 7.38 (2H, m)
Example 24
(4aS,6R,8aS)-6H-Benzofuro[3a,3,2-ef][2]benzazepin-6-ol,
4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-, hydrogen sulfate
(ester)
##STR00092##
[0296] Chlorsulfonic acid (0.16 g, 1.39 mmol) was added to dry
pyridine (1 ml) preheated to 70-80.degree. C. and stirred at the
same temperature for 30 min. A solution of galantamine (0.20 g,
0.70 mmol) in dry pyridine (1 ml) was added drop wise and the
mixture was stirred overnight at room temperature with the
formation of a precipitate. MeOH/H.sub.2O 1:1 (5 ml) was added and
the resulting clear solution was stirred for further 30 min.
Volatiles were rotoevaporated and another portion of MeOH (5 ml)
was added. The resulting fine precipitate was filtered to give
(0.21 g, yield 82%, HPLC >99%) of product as a white powder.
[0297] IR: 1700.59, 1652.92, 1623.93, 1617.01, 1510.15, 1475.31,
1443.53, 1299.82, 1282.40, 1266.98, 1242.70, 1217.83, 1197.48,
1155.3, 1092.40, 1070.97, 1053.15, 1023.70, 1007.21, 984.45.
Example 25
General Procedure 1
[0298] To the solution of (-)-galantamine hydrobromide (1.0 mole)
and triethyl amine (4.0 mol) in DCM (30 mL), DMAP (0.5 mol) was
added followed by respective acid chloride or acid anhydride (1.2
mol). The mixture was stirred overnight at room temperature under
argon. The reaction mixture was washed with 10% NaHCO.sub.3 and
brine, dried (Na.sub.2SO.sub.4) and concentrated. The crude
compound obtained was purified by column chromatography or
recrystallization to give the pure product.
##STR00093## ##STR00094##
Using this procedure the following compounds were obtained:
[0299] O-Benzoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, benzoate (ester)); yield: 78%
[0300] O-3,4-Dichlorobenzoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 3,4-dichlorobenzoate (ester));
off-white solid; mp. 69-70.degree. C.
[0301] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. (ppm) 8.02 (d,
J=1.88 Hz, 1H), 7.81 (dd, J=1.88 Hz, J=8.38 Hz, 1H), 7.38 (d,
J=8.32 Hz, 1H), 6.62 (d, J=8.18 Hz, 1H), 6.52 (d, J=8.18 Hz, 1H),
6.32 (d, J=10.34 Hz, 1H), 5.89-5.97 (m, 1H), 5.51 (t, J=4.43 Hz,
1H), 4.58 (s, 1H), 4.07 (d, J=15.16 Hz, 1H), 3.18 (s, 3H), 3.61 (d,
J=15.16 Hz, 1H), 3.21-3.45 (m, 1H), 2.96-3.05 (m, 1H), 2.66-2.76
(m, 1H), 2.34 (s, 3H), 2.0-2.19 (m, 2H), 1.51-1.59 (m, 1H).
[0302] O-4-Methoxybenzoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 4-methoxybenzoate (ester));
off-white solid; mp. 183-184.degree. C.
[0303] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. (ppm) 8.01 (d,
J=9.0 Hz, 2H), 8.56 (d, J=8.86 Hz, 2H), 6.69 (d, J=8.18 Hz, 1H),
6.58 (d, J=8.2 Hz, 1H), 6.35 (d, J=10.2 Hz, 1H), 6.0-6.07 (m, 1H),
5.56 (t, J=4.49 Hz, 1H), 4.66 (s, 1H), 4.15 (d, J=15.18 Hz, 1H),
3.89 (s, 3H), 3.84 (s, 3H), 3.68 (d, J=15.18 Hz, 1H), 3.29-3.53 (m,
1H), 3.04-3.12 (m, 1H), 2.73-2.81 (m, 1H), 2.41 (s, 3H), 2.08-2.26
(m, 2H), 1.58-1.66 (m, 1H).
[0304] O-4-Methylbenzoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 4-methylbenzoate (ester)); off-white
solid; mp. 71-72.degree. C.
[0305] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. (ppm) 7.94 (d,
J=8.18 Hz, 2H), 7.17 (d, J=8.06 Hz, 2H), 6.69 (d, J=8.18 Hz, 1H),
6.58 (d, J=8.2 Hz, 1H), 6.35 (d, J=9.52 Hz, 1H), 6.0-6.08 (m, 1H),
5.57 (t, J=4.43 Hz, 1H), 4.66 (s, 1H), 4.17 (d, J=15.18 Hz, 1H),
3.89 (s, 3H), 3.70 (d, J=15.18 Hz, 1H), 3.31-3.43 (m, 1H),
3.06-3.13 (m, 1H), 2.74-2.83 (m, 1H), 2.42 (s, 3H), 2.38 (s, 3H),
2.08-2.26 (m, 2H), 1.59-1.67 (m, 1H).
[0306] O-4-Chlorobenzoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 4-chlorobenzoate (ester)); off-white
solid; mp. 72-74.degree. C.
[0307] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. (ppm) 7.91 (d,
J=8.74 Hz, 2H), 7.27 (d, J=8.72 Hz, 2H), 6.62 (d, J=8.2 Hz, 1H),
6.52 (d, J=8.2 Hz, 1H), 6.30 (d, J=10.34 Hz, 1H), 5.92-6.0 (m, 1H),
5.5 (t, J=4.36 Hz, 1H), 4.59 (s, 1H), 4.09 (d, J=15.18 Hz, 1H),
3.82 (s, 3H), 3.63 (d, J=15.18 Hz, 1H), 3.23-3.46 (m, 1H),
2.99-3.06 (m, 1H), 2.66-2.76 (m, 1H), 2.35 (s, 3H), 2.0-2.2 (m,
2H), 1.52-1.6 (m, 1H).
[0308] O-2-Thenoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, thiophene-2-carboxylate (ester));
off-white solid; mp. 115-116.degree. C.
[0309] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. (ppm) 7.78 (dd,
J=1.2 Hz, J=3.8 Hz, 1H), 7.51 (dd, J=1.34 Hz, J=4.96 Hz, 1H), 7.04
(dd, J=3.76 Hz, J=4.98 Hz, 1H), 6.69 (d, J=8.18 Hz, 1H), 6.59 (d,
J=8.04 Hz, 1H), 6.35 (d, J=10.2 Hz, 1H), 6.02 (dd, J=4.7 Hz, J=10.2
Hz, 1H), 5.54 (t, J=4.49 Hz, 1H), 4.63 (s, 1H), 4.18 (d, J=15.02
Hz, 1H), 3.87 (s, 3H), 3.71 (d, J=15.18 Hz, 1H), 3.31-3.5 (m, 1H),
3.07-3.14 (m, 1H), 2.73-2.83 (m, 1H), 2.42 (s, 3H), 2.04-2.26 (m,
2H), 1.6-1.68 (m, 1H).
[0310] O-5-Chloro-2-thenoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 5-chlorothiophene-2-carboxylate
(ester)); off-white solid; mp. 58-59.degree. C.
[0311] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. (ppm) 7.5 (d,
J=4.04 Hz, 1H), 7.80 (d, J=4.02 Hz, 1H), 6.62 (d, J=8.04 Hz, 1H),
6.52 (d, J=8.06 Hz, 1H), 6.31 (d, J=10.2 Hz, 1H), 5.92 (dd, J=4.57
Hz, J=10.2 Hz, 1H), 5.45 (t, J=4.36 Hz, 1H), 4.56 (s, 1H), 4.08 (d,
J=15.16 Hz, 1H), 3.81 (s, 3H), 3.61 (d, J=15.18 Hz, 1H), 3.21-3.34
(m, 1H), 2.97-3.04 (m, 1H), 2.64-2.74 (m, 1H), 2.34 (s, 3H),
1.97-2.19 (m, 2H), 1.5-1.57 (m, 1H).
Example 26
General Procedure 2
[0312] To the solution of the corresponding acid acid (13.87 g,
135.8 mmol) in DCM (250 mL) was added DCC (33.62 g, 162.9 mmol)
followed by DMAP (3.32 g, 27.15 mmol), reaction mixture was stirred
for additional 30 minutes at room temperature. To this
(-)-galantamine hydrobromide (10.0 g, 27.15 mmol) and triethyl
amine (4.6 mL, 32.59 mmol) was added, the mixture was stirred
overnight at room temperature under argon. The precipitated DCHU
was removed by filtration and the filtrate was evaporated. The
additional DCHU was removed by subsequent trituration with cold
ethyl acetate and filtration. The ethyl acetate solution was
roto-evaporated and the crude product obtained was purified by
column chromatography to give the desired product.
[0313] 2-Methyl-butanoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 2-methyl-butanoate (ester)) was
obtained in 53% yield as a solid using the general procedure 2.
[0314] The same product, identical in every respect (HPLC, m.p.,
.sup.1H-NMR), was also obtained in 58% yield using the general
procedure 1.
[0315] 2-Methyl-propanoyl-galantamine
(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro-
[3a,3,2-ef][2]benzazepin-6-ol, 2-methyl-propanoate (ester)) was
obtained in 63% yield as a solid using the general procedure 1.
2-Methyl-propanoyl-galantamine hydrochloride salt
[0316] To the solution of 2-methyl-propanoyl-galantamine (150 mg,
0.43 mmol) in ethyl acetate (5 mL), ethyl acetate saturated with
HCl (5 mL) was added slowly with stirring at 0.degree. C. The
reaction mixture was stirred at room temperature for 1 h. Solvent
was evaporated and the residue obtained was washed with dry ether
and was dried under high vacuum to give 164 mg (97%) of desired
product as an off-white solid.
[0317] Anal. calcd for C.sub.21H.sub.27NO.sub.4 (1.5HCl): C, 61.2;
H, 6.97; N, 3.40. Found: C, 61.62; H, 6.95; N, 3.91.
2-Methyl-propanoyl-galantamine citric acid salt
[0318] To the solution of 2-methyl-propanoyl-galantamine (150 mg,
0.43 mmol) in methanol (5 mL), a solution of citric acid in
methanol (5 mL) was added slowly with stirring at room temperature.
The reaction mixture was stirred at room temperature for 1 h.
Solvent was evaporated and the residue obtained was precipitated
from methanol-diethyl ether and was dried under high vacuum to give
187 mg (81%) of desired product as a off-white solid.
[0319] Anal. calcd for C.sub.27H.sub.35NO.sub.11 (1.0H.sub.2O): C,
57.14; H, 6.57; N, 2.47. Found: C, 57.43; H, 6.48; N, 2.53.
Example 27
General Procedure 3
[0320] To a stirred solution of (-)-galantamine hydrobromide (1.10
mmol) in pyridine (6 mL) at 0.degree. C. under nitrogen, the
corresponding acid chloride (2.2 mmol) was added and the mixture
was stirred until a TLC showed the reaction to be complete. Then
CH.sub.2CL.sub.2 (10 mL) and water (10 mL) were added and stirring
was continued for 30 min. The organic layer was separated, washed
with water (2.times.10 mL), dried over anhydrous MgSO.sub.4 and the
solvent removed. The residue was purified by flash chromatography
giving the product identical in all respects to
O-Benzoyl-galantamine.
Example 28
Synthesis of R1-pyridinoyl-galantamine
[0321] In addition to the examples provided above, the following
compounds were prepared by the described general procedures:
TABLE-US-00007 TABLE 6 MF MW Substituent R1
C.sub.21H.sub.25NO.sub.4 355.43 cyclopropanecarboxylate
C.sub.24H.sub.23Cl.sub.2NO.sub.4 460.3 3,4-dichlorobenzoate
C.sub.28H.sub.33NO.sub.4 447.57 4-tert-butylbenzoate
C.sub.25H.sub.23ClF.sub.3NO.sub.4 493.91
4-chloro-3-(trifluoromethyl)benzoate
C.sub.25H.sub.23F.sub.3N.sub.2O.sub.6 504.46
4-nitro-3-(trifluoromethyl)benzoate C.sub.25H.sub.24F.sub.3NO.sub.4
459.47 4-(trifluoromethyl)benzoate C.sub.24H.sub.23Cl.sub.2NO.sub.4
460.36 2,4-dichlorobenzoate C.sub.24H.sub.24N.sub.2O.sub.6 436.47
4-nitrobenzoate C.sub.24H.sub.24ClNO.sub.4 425.92 3-chlorobenzoate
C.sub.25H.sub.24F.sub.3NO.sub.4 459.47 3-(trifluoromethyl)benzoate
C.sub.24H.sub.24N.sub.2O.sub.6 436.47 3-nitrobenzoate
C.sub.24H.sub.23Cl.sub.2NO.sub.4 460.36 3,5-dichlorobenzoate
C.sub.26H.sub.30N.sub.2O.sub.4 434.54 3-(dimethylamino)benzoate
C.sub.25H.sub.27NO.sub.4 405.50 3-methylbenzoate
C.sub.24H.sub.24ClNO.sub.4 425.92 2-chlorobenzoate
C.sub.24H.sub.23F.sub.2NO.sub.4 427.45 2,4-difluorobenzoate
C.sub.24H.sub.23Cl.sub.2NO.sub.4 460.36 2,5-dichlorobenzoate
C.sub.24H.sub.24FNO.sub.4 409.46 4-fluorobenzoate
C.sub.26H.sub.30N.sub.2O.sub.4 434.54 4-(dimethylamino)benzoate
C.sub.24H.sub.26N.sub.2O.sub.4 406.49 4-aminobenzoate
C.sub.27H.sub.32N.sub.2O.sub.4 448.57
4-(dimethylamino)-3-methylbenzoate C.sub.25H.sub.25NO.sub.6 435.48
2H-1,3-benzodioxole-5-carboxylate C.sub.26H.sub.27NO.sub.5 433.51
4-acetylbenzoate C.sub.22H.sub.23NO.sub.4S 397.50
thiophene-3-carboxylate C.sub.21H.sub.23N.sub.3O.sub.4 381.44
1H-imidazole-5-carboxylate C.sub.21H.sub.22N.sub.2O.sub.5 382.42
1,3-oxazole-5-carboxylate C.sub.21H.sub.22N.sub.2O.sub.4S 398.48
1,3-thiazole-5-carboxylate C.sub.21H.sub.22N.sub.2O.sub.4S 398.48
1,3-thiazole-2-carboxylate C.sub.26H.sub.27NO.sub.6 449.51
2-(acetyloxy)benzoate
Example 29
Preparation of Mouse Brain Homogenate
[0322] The brain is removed from the animal (mouse), shock frozen
in liquid nitrogen and stored at minus 80.degree. C. until use.
Before preparing the extract, the frozen brain is thawed on ice and
the weight is determined. Ice cold buffer (130 mM NaCl, 5 mM KCl,
2.5 mM CaCl.sub.2, 1 mM MgCl.sub.2, 5 mM Glucose, 5 mM HEPES, pH
7.4) is added to the thawed mouse brain (1:4, weight to volume,
resulting in a 20% brain homogenate). The tissue is then
homogenized in a potter homogeniser on ice, moving the piston up
and down 11 times at 240 rpm. The freshly prepared mouse brain
homogenate is divided into aliquots.
Example 30
Procedure for Measuring the Inhibition of Brain Esterase
Results Shown as FIG. 1
[0323] A modified Ellmann's esterase test is used. Briefly the
method relies on the cleavage of the substrate acetylthiocholine to
acetate and thiocholine. The latter reacts with DTNB
(5,5'-Dithiobis-(2-nitro-benzoicacid) to a yellow compound, which
can be quantified spectrometrically. The incubation buffer contains
51 mmol/l sodium phosphate buffer and 0.05% Tween 20, at pH 7.2 and
is supplemented with 100 mg/l DTNB, and 0.2% mouse brain homogenate
(prepared as described in Example 29). The compound to be
investigated is added to the desired concentration. The mixture is
brought to 37.degree. C. and the reaction is started by addition of
200 .mu.M acetylthiocholine. A.sub.405 is measured in 1 s intervals
in a microplate reader for 40 s. The linear parts of the
absorption-time-curves represent the starting speed of the
enzymatic reaction and are used for the calculation of the reaction
speed. The slope of the curve corresponds to the reaction speed.
The inhibition is expressed as percent of the non inhibited
reaction according to following equation:
%
Inhibition=100*(1-(Slope.sub.inhibited/Slope.sub.noninhibited)).
Example 31
Procedure for Determination of Prodrug Cleavage in Mouse Brain
Homogenate
[0324] To aliquots of mouse brain homogenate as prepared according
to Example 13 pro-galantamine derivatives are added and adjusted to
a final concentration of 10 .mu.M of the prodrug. At the end of the
incubation time, 12 .mu.l 0.1 M NaOH and 100 .mu.l saturated KCl
are added to the 0.1 ml reaction mixture and mixed thoroughly. The
remaining prodrug and the released galantamine are extracted by 200
.mu.l toluene. The toluene extraction step is repeated twice using
150 .mu.l toluene and the obtained extracts are pooled, dried,
dissolved in 50% Methanol and used for subsequent HPLC
analysis.
Example 32
Investigation of the Allosteric Modulation Effect of Drug
Candidates on Nicotinic Acetylcholine Receptors (nAChRs) Expressed
in HEK-293 Cells by Electrophysiology
Results Shown as FIG. 4
[0325] Single HEK-293 cells expressing either human
.alpha.4.beta.2, human .alpha.3.beta.4, or chimeric chicken
.alpha.7 (with mouse 5HT.sub.3) nAChR were plated on
fibronectin-coated cover slips for 3 days before measurement. The
selected nAChR containing cells were placed in the recording bath,
filled with extracellular buffer (145 mM NaCl, 5 mM KCl, 1 mM
MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM D-glucose, 10 mM HEPES, pH 7.3,
approximately 300 mOsm). Patch-clamp system consisted of an
inverted microscope (Zeiss, Germany), computer-controlled
patch-clamp amplifier with PatchMaster software (HEKA, Germany),
tubing perfusion system (ALA, USA) together with a U-tube
applicator (IMM, Germany) and dual micromanipulators. The patch
pipettes were pulled from fire-polished, 100 mm long and 1.5 mm
width, single borosilicate glass capillaries (WPI, Germany). A
programmable puller (Sutter, USA) was used to prepare a twin pair
of ready for use pipettes. Each patch pipette (resistance 4-8
M.OMEGA.) was used only once. Pipettes were filled with an internal
buffer (140 mM CsCl, 11 mM EGTA, 10 mM HEPES, 2 mM MgCl.sub.2, pH
7.3, approximately 300 mOsm) and connected to the working
electrode. Working and reference electrodes for experiments were
made from daily renewed, freshly chlorinated silver wire (40
mm.times.0.4 mm) and were connected to a headstage circuit of the
patch-clamp amplifier. Patching was done using rectangular test
pulses with an amplitude of -1 mV and a duration of 20 ms.
Immediately after formation of the gigaseal the holding potential
of -70 mV was applied to the patch electrode and whole-cell
recordings were established by using negative pressure pulses. All
necessary compensations for fast and slow membrane capacitance and
serial resistance transients were automatically set within the
PatchMaster software. Whole-cell currents were evoked by the
application of nicotine at the EC.sub.50 for each appropriate nAChR
subtype (.alpha.4.beta.2 and .alpha.3.beta.4 EC.sub.50=30 .mu.M,
chimeric .alpha.7 EC.sub.50=3 .mu.M). To evaluate an allosteric
potentiating ligand (APL) effect of selected compounds on each
subtype of nAChR, they were added to stimulating nicotine solutions
at the following concentrations: 1, 5, 10, 50, 100, 500, 1,000,
5,000 and 10,000 nM, and solutions were applied to the cell surface
during 500 ms pulses through the U-tube, and then corresponding
currents, digitized to 10 kHz, were recorded for 10 s. Consecutive
current stimulations were done with a 2 min interval to avoid nAChR
desensibilisation and to ensure full exchange of stimulating
solutions. The averaged peak amplitudes of the currents, measured
in the presence of selected compound concentrations, were compared
with those determined in the absence of compounds (control) and
they were calculated as % of control. The measurements of an APL
effect of particular compound were repeated on a minimum of five
cells to obtain the mean values+/-SD. Mean values of the observed
APL effect, which did not exceed 15% were treated as insignificant.
To present a concentration-dependent APL effect of particular
compound, the corresponding % of control values+/-SD were plotted
against the concentrations used.
Example 33
Pharmacokinetics of Pro-Galantamine Gln-1062 (3 Mg/Kg) in the
Mouse
Results Shown as FIG. 5
Study Objective
[0326] Determination of the pharmacokinetic profiles of Gln-1062
and its cleavage product galantamine in blood and brain. Determine
the brain-to-blood concentration ratios of Gln-1062 and its
cleavage product galantamine, and assess the blood-brain barrier
penetration capacities.
Study Plan
Bioanalysis
[0327] Analytical method for estimation of Gln-1062 was evaluated
for its linearity, precision & accuracy and recovery in SAM
blood and brain homogenate using LC/MS/MS.
LC/MS/MS Parameters
[0328] The parameters of chromatographic conditions and extraction
conditions for the Gln-1062 and Galantamine analysis were
[0329] Chromatographic parameters: [0330] Column: Phenomenex
Synergi, Polar-RP 80 A, C18, 75.times.2.0 mm, 4.mu. [0331] Mobile
Phase [0332] Mobile Phase Buffer: 40 mM Ammonium Formate, pH 3.5
[0333] Aqueous Reservoir (A): 10% Buffer, 90% Water [0334] Organic
Reservoir (B): 10% Buffer, 90% Acetonitrile [0335] Flow rate: 0.450
mL/min [0336] Gradient Programme:
TABLE-US-00008 [0336] Gradient Time (min) Curve % A % B 0 1 100 0
1.2 1 60 40 3 1 0 100 3.1 1 100 0 5 1 100 0
[0337] Divert valve time schedule:
TABLE-US-00009 [0337] Divert Valve Time (min) Waste MS 0 x 1.2 x
4.5 x
[0338] Run time: 5.0 min [0339] Column oven temperature: Ambient
[0340] Auto sampler temperature: Ambient [0341] Auto sampler Wash:
Water:acetonitrile:isopropanol with 0.2% formic acid, 1:1:1(v/v/v)
[0342] Retention time: Gln-1062: 3.33.+-.0.05 min. [0343]
Galantamine: 2.44.+-.0.05 min [0344] Metoprolol: 2.80.+-.0.05 min.
[0345] Mass Parameters (API 3200): [0346] Mode: MRM [0347]
Polarity: Positive [0348] Ion source: Turbo spray [0349] Analyte:
Gln-1062 (Q1 Mass 392.4; Q3 Mass 213.2) [0350] Galantamine (Q1 Mass
288.3; Q3 Mass 213.1) [0351] ISTD: Metoprolol (Q1 Mass 268.4; Q3
Mass 116.2)
[0352] Source/Gas Parameters:
TABLE-US-00010 Curtain gas (CUR) 10 Collision gas (Collision
associated Dissociation) CAD 5 Ion Spray Voltage (IS) 5500 V
Temperature (TEM) 575.degree. C. GS1 55 GS2 45 Ihe ON
[0353] Compound Parameters:
TABLE-US-00011 Parameter Galantamine Metoprolol Gln-1062
Declustering Potential (V) 45 35 50 Entrance Potential (V) 10 10 10
Collision Cell Entrance Potential 20 20 20.00 (V) Collision Energy
(eV) 32 26 32 Collision Cell Exit Potential (V) 4.5 2 5 Dwell Time
(milliSec) 200 200 200
[0354] Extraction Procedure:
[0355] A. Preparation of STD, QC and Study Samples
##STR00095##
[0356] B. Preparation of Calibration Curve Standards for
Recovery
##STR00096##
Method Evaluation
[0357] This method was evaluated for linearity, precision &
accuracy and recovery of Gln-1062 in SAM blood and brain
homogenate.
[0358] A. Linearity, Precision & Accuracy [0359] A single
standard curve and six replicates each of three quality control
(QC) levels (18 total QCs) were extracted and analyzed. The
linearity of the calibration curve was determined by a weighed
least square regression analysis. [0360] Acceptance Criteria [0361]
i. At least six out of nine standards must have an accuracy of
.+-.15% from nominal, except at the lower limit of quantitation
(LLOQ) where .+-.20% is acceptable. [0362] ii. Two-thirds of the
batch QCs and at least half of the QCs at each level must have a
calculated accuracy of .+-.15% from nominal. [0363] iii.
Intra-assay Mean Precision and Accuracy [0364] 1. Four out of six
QCs must be available to determine accuracy and precision. [0365]
2. The intra-assay coefficient of variation (% CV) of each QC level
must not exceed 15% and the accuracy of the mean value for each
validation to be accepted. [0366] Gln-1062 linearity, precision and
accuracy in blood and brain homogenate matrices were evaluated.
[0367] B. Recovery
[0368] The recovery of Gln-1062 from the blood and brain homogenate
matrices were also evaluated.
[0369] Recovery was determined by quantifying the concentration of
the analytes in extracted matrix QC samples with a standard curve
prepared in post-extract (blank extracts) sample matrix as
described above in section B of the extraction procedure, entitled
"Preparation of Calibration Curve Standards for Recovery."
[0370] Gln-1062 recovery in blood and brain homogenate matrices
were evaluated.
Animal Study
[0371] Study Design
TABLE-US-00012 Dose Dose No. of Dose Conc. Volume Animals for
Sample time Animal Test item (mg/kg) (mg/mL) (mL/kg) Dose route
each time point points (hr) Male SAM Gln-1062 3 0.2 15 i.v, bolus 3
Predose, 0.05, 0.10, 25-33 gm (tail vein) 0.17, 0.33, 0.50, 0.83,
1.33, 2.0 and 4.0
Sample Collection
Collection of Blood:
[0372] Blood samples were collected from the retro-orbital plexus.
0.5 ml of blood was collected into a pre-labeled polypropylene
micro centrifuge tube containing sodium citrate as the
anticoagulant, and kept on ice.
[0373] Blood was mixed gently with anticoagulant and an aliquot of
500 .mu.L of blood sample immediately precipitated as described
above in section A of the extraction procedure, entitled
"Preparation of STD, QC and Study Samples." Remaining volume of
blood sample at each time point frozen on dry ice.
[0374] All the blood samples were transferred to analytical
department and frozen at -80.+-.10.degree. C. until analysis.
Collection of Brain:
[0375] Immediately after blood withdrawal, brain was perfused with
phosphate buffer saline (pH 7.4), removed and frozen on dry
ice.
[0376] All the brain samples were transferred to analytical
department and frozen at -80.degree. C. until analysis.
Brain Homogenate Preparation:
[0377] Brain samples were thawed on ice and weighed. n appropriate
volume ice cold homogenizing media (methanol:water::20:80,v/v)
added. n ice, homogenized the brain sample with the polytron
homogenizer and make up the volume with homogenizing media to get 1
gm of brain per 4 mL of homogenate. After homogenizing, immediately
freezed the brain homogenate samples at -80.degree. C. until
analysis.
Example 34
Behavioural Index for Gastro-Intestinal Side Effects in Ferrets
Following Application of Galantamine and Several
R1-Pro-Galantamines, Respectively
Results Shown in FIG. 6
Test System
[0378] Fourteen adult male Putoris furo ferrets (Marshall
BioResources (North Rose, USA)), weighting 750-1000 grams on the
day of experimentation were used in the present study. In agreement
with the sponsor four out of the fourteen animals were included in
two experimental groups, ferrets #1, 2, 3 and 4).
Animal Housing
[0379] The acclimatization of the animals lasted at least 5 days.
At receipt, animals were collectively housed in cages at
Syncrosome's premises. They had free access to food and drinking
water ad libitum.
Test Item and Reference Compound
[0380] During this study, one reference compound (Galantamine) and
one
[0381] Galantos candidate compound (GLN979) were tested at two
doses each. Both compounds were administered I.P. at doses and
concentrations.
Details of the different compound shipments are presented in Table
7.
TABLE-US-00013 TABLE 7 Receipt Nov. 11.sup.th, Compound 2007 Nov.,
the 21.sup.st, 2007 Nov., the 29.sup.th, 2007 Galantamine 48.0 mg
256.0 mg (One vial) -- GLN979 48.0 mg -- 262.6 mg 2- U.I: 7.3 g +
7.3 g (Two .apprxeq.4.7 g + .apprxeq.5.0 g Hydroxypropyl- (One
vial) vials) .beta.- cyclodextrin NaCl U.I: 3.0 g (One vial) --
(One vial)
[0382] As requested by the sponsor, the same vehicle (15%
2-Hydroxypropyl-.beta.-cyclodextrin/96 mM NaCl) was used for both
Galantamine and GLN979 preparation. A detailed solubilization
protocol was sent by mail by Galantos to Syncrosome and received on
Nov. 5, 2007.
Test Compound (GLN979)
TABLE-US-00014 [0383] Nature GLN979. Molar mass U.I. Administration
dose 20 and 40 mg/kg B.W. Administration route I.P. Vehicle 15%
2-Hydroxypropyl-.beta.-cyclodextrin/96 mM NaCl.
Reference Compound (Galantamine)
TABLE-US-00015 [0384] Nature Galantamine Molar mass U.I.
Administration dose 3 and 20 mg/kg B.W. Administration route I.P.
Vehicle 15% 2-Hydroxypropyl-.beta.-cyclodextrin/96 mM NaCl.
I.P. Administrations
[0385] For the 4 experimental groups, the administrations were
performed in unanaesthetized animals through the I.P. route at
T.sub.0.
Emesis Test
[0386] After I.P. administration of the compound solution, the
animals were continuously observed by a trained technician for four
hours. During that period, the number of episodes of vomiting
(series of retches leading to the expulsion of part of the
gastro-intestinal content) were recorded.
Behavioural Observation
[0387] During the 4 hours-observation period, several side-effects
(salivation, shivering, respiratory problems and diarrhea) were
also observed. For each of these behaviours, a scoring method was
determined with the sponsor. Depending on its severity, each
parameter were quantified as: [0388] "None" (None): Behaviour not
observed. [0389] "Moderate" (Mod.): Behaviour observed with a low
frequency and/or a low intensity. [0390] "Intense" (Int.):
Behaviour observed frequently and/or continuously and/or at a high
intensity.
Inclusion Criteria
[0391] All the animals receiving I.P. administration of the
reference or the test compound were included in the study
regardless of the pattern of both their emetic responses and
behaviors.
Example 35
Reversal from Scopolamine-Induced Amnesia in Mice
Results Shown as FIG. 6
Drug Preparation
[0392] Gln 1062, Gln 0979 and galantamine were dissolved in
15%-hydroxypropyl-.beta.-cyclodextrin in 96 mM NaCl (isotonic)
supplied by the sponsor. Gln 1062 and Gln 0979 were used in
concentrations of 0.01, 0.03, 0.1 and 0.2 mg/ml, which when given
in a volume of 10 ml/kg result in doses of 0.1, 0.3, 1 and 2 mg/kg
i.p., respectively. Galantamine was used in concentrations of 0.03,
0.1, 0.2 and 0.5 mg/ml, which when given in a volume of 10 ml/kg
result in doses of 0.3, 1, 2 and 5 mg/kg i.p., respectively.
[0393] Control animals received
15%-hydroxypropyl-.beta.-cyclodextrin in 96 mM NaCl injection as a
vehicle.
[0394] Nicotine ((-)-Nicotine hydrogen tartrate salt, Sigma,
France), scopolamine
[0395] (-(-)scopolamine hydrochloride, Sigma, France) were
dissolved in saline (0.9% NaCl, Aguettant, France) at the
concentration of 0.04 and 0.1 mg/ml, respectively. They were
administrated at a dosage volume of 10 ml/kg to achieve doses of
0.4 and 1 mg/kg, respectively.
Test Animals
[0396] Four to five week old male CD-1 mice (Janvier; Le Genest St
Isle--France) were used for the study.
[0397] They were group-housed (10 mice per cage) and maintained in
a room with controlled temperature (21-22.degree. C.) and a
reversed light-dark cycle (12 h/12 h; lights on: 17:30-05:30;
lights off: 05:30-17:30) with food and water available ad
libitum.
Experimental Design
[0398] The potential cognitive enhancing property of Gln 1062, Gln
0979 and galantamine were evaluated in scopolamine-treated mice in
the T-maze alternation model under the same experimental
conditions. Both Gln 1062 and Gln 0979 were tested in doses of 0.1,
0.3, 1 and 2 mg/kg i.p. Galantamine was tested in doses 0.1, 0.3,
1, 2 and 5 mg/kg i.p. Nicotine was tested in a dose of 0.4 mg/kg
i.p. All these compounds were administrated immediately after the
injection of 1 mg/kg i.p. scopolamine (20 min prior to the T-maze
trial) used to induce memory deficit.
[0399] Memory performance was assessed by the percentage of
spontaneous alternation in the T-maze. The number of alternation in
saline-injected mice was used as the base level of unaltered memory
performance.
[0400] Mice were housed 10 per cage. Each mouse in a cage was
randomly assigned by a unique number (1 to 10) written on the tail
with permanent ink.
[0401] Gln 1062, Gln 0979 and galantamine were tested separately in
three set of experiments with different animals. Each set of
experiments was divided in series of daily experiments that always
comprises at least one representative of each of Saline/vehicle,
Scopolamine/vehicle and Scopolamine/Nicotine (0.4 mg/kg)
groups.
Measurement
[0402] The T-maze apparatus was made of gray Plexiglas with a main
stem (55 cm long.times.10 cm wide.times.20 cm high) and two arms
(30 cm long.times.10 cm wide.times.20 cm high) positioned at 90
degree angle relative to the left and right of the main stem. A
start box (15 cm long.times.10 cm wide) was separated from the main
stem by a guillotine door. Horizontal doors were present to close
off specific arms during the force choice alternation task.
[0403] The experimental protocol consists of one single session,
which starts with 1 "forced-choice" trial, followed by 14
"free-choice" trials. In the first "forced-choice" trial, the
animal is confined 5 s in the start arm and then released while
either the left or right goal arm is blocked by a horizontal door.
After the mouse is released, it will negotiate the maze and
eventually enter the open goal arm, and return to the start
position. Immediately after the return of the animal to the start
position, the closed goal door is opened and the animal is now free
to choose between the left and right goal arm ("free choice
trials"). The animal is considered as entered when it places its
four paws in the arm. A session is terminated and the animal is
removed from the maze as soon as 14 free-choice trials have been
performed or 10 min have elapsed, whatever event occurs first. The
average duration of a trial is 6 min.
[0404] The apparatus is cleaned between each animal using
alcohol(70.degree.). Urine and feces are removed from the maze.
[0405] During the trials, animal handling and the visibility of the
operator are minimized as much as possible.
[0406] The percentage of alternation over the 14 free-choice trials
was determined for each mouse and was used as an index of working
memory performance. This percentage was defined as entry in a
different arm of the T-maze over successive trials (i.e.,
left-right-left-right, etc).
Statistical Analysis
[0407] Analysis of variance (ANOVA) was performed on the result
data. Fisher's Protected Least Significant Difference was used for
pairwise comparisons. p value.ltoreq.0.05 were considered
significant. The drug induced reversion of scopolamine-induced
memory deficit was calculated by setting the respective response of
the saline/vehicle group as 100% and the scopolamine/vehicle group
as 0% reversion.
[0408] In order to determine the ED.sub.50 for each drug, the
recovery performance was plotted following a sigmoidal
dose-response model (graphpad software). ED50 was read from the
curve fitting table and represents the effective dose associated
with 50% of response.
[0409] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of any appended claims.
All figures, tables, and appendices, as well as publications,
patents, and patent applications, cited herein are hereby
incorporated by reference in their entirety for all purposes.
* * * * *